U.S. patent application number 11/920881 was filed with the patent office on 2009-09-03 for north-2'-deoxy-methanocarbathymidines as antiviral agents against poxviruses.
Invention is credited to Victor E. Marquez, Christopher K. Tseng.
Application Number | 20090221523 11/920881 |
Document ID | / |
Family ID | 37452982 |
Filed Date | 2009-09-03 |
United States Patent
Application |
20090221523 |
Kind Code |
A1 |
Tseng; Christopher K. ; et
al. |
September 3, 2009 |
North-2'-deoxy-methanocarbathymidines as antiviral agents against
poxviruses
Abstract
A method for the prevention or treatment of poxvirus infection
by administering an effective amount of an antiviral agent
comprising cyclopropanated carbocyclic 2'-deoxynucleoside to an
individual in need thereof is provided.
Inventors: |
Tseng; Christopher K.;
(Burtonsville, MD) ; Marquez; Victor E.;
(Montgomery Village, MD) |
Correspondence
Address: |
KNOBBE, MARTENS, OLSON & BEAR, LLP
2040 MAIN STREET, FOURTEENTH FLOOR
IRVINE
CA
92614
US
|
Family ID: |
37452982 |
Appl. No.: |
11/920881 |
Filed: |
May 25, 2006 |
PCT Filed: |
May 25, 2006 |
PCT NO: |
PCT/US2006/020894 |
371 Date: |
April 3, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60684811 |
May 25, 2005 |
|
|
|
Current U.S.
Class: |
514/50 |
Current CPC
Class: |
A61P 31/20 20180101;
A61K 31/7072 20130101 |
Class at
Publication: |
514/50 |
International
Class: |
A61K 31/7072 20060101
A61K031/7072; A61P 31/20 20060101 A61P031/20 |
Claims
1. A method of treating a poxvirus infection in a patient in need
thereof, comprising the step of: administering to the patient, in a
pharmaceutically acceptable carrier, an effective antiviral amount
of a compound having the formula: ##STR00019## wherein R.sub.1 is
selected from the group consisting of a substituted or
unsubstituted adenine moiety, a substituted or unsubstituted
thymine moiety, a substituted or unsubstituted cytosine moiety, a
substituted or unsubstituted uracil moiety, and a substituted or
unsubstituted guanine moiety.
2. A method of treating a poxvirus infection in a patient in need
thereof, comprising the step of: administering, in a
pharmaceutically acceptable carrier, to said mammal an effective
antiviral amount of a compound having the formula: ##STR00020##
wherein R is selected from the group consisting of hydrogen,
fluorine, chlorine, bromine, iodine, hydroxyl, --CH.sub.2--X.sup.2,
--CH.dbd.CH--X.sup.2, and --C.dbd.C--X.sup.2, X.sup.1 is
independently selected from the group consisting of hydrogen,
fluorine, chlorine, bromine, and iodine; and X.sup.2 is
independently selected from the group consisting of hydrogen,
fluorine, chlorine, bromine, and iodine.
3. The method of claim 2, wherein the compound is of the formula:
##STR00021##
4. The method of claim 3, wherein X.sup.1 is hydrogen.
5. The method of claim 4, wherein R is selected from the group
consisting of fluorine, bromine, and iodine.
6. The method of claim 4, wherein R is methyl.
7. The method of claim 2, wherein the poxvirus is smallpox
virus.
8. The method of claim 2, wherein the poxvirus is selected from the
group consisting of vaccinia, monkeypox, cowpox, rabbitpox, raccoon
pox, tatera pox, buffalopox, and camelpox.
9. The method of claim 2, wherein the poxvirus is selected from the
group consisting of fowl pox, canary pox, goat pox, sheep pox,
lumpy skin disease, myxoma, hare fibroma, orf, pseudo-cowpox,
swinepox, molluscum contagiosum, tanapox, yaba, and mousepox.
10. The method of claim 2, wherein the patient is a human.
11. The method of claim 10, wherein the effective antiviral amount
is from about 300 mg per day to about 15,000 mg per day.
12. A method of treating a poxvirus infection in a patient in need
thereof, comprising the step of administering to the individual an
effective antiviral amount of North-methanocarbathymidine
triphosphate.
13. The method of claim 12, wherein the poxvirus is smallpox
virus.
14. The method of claim 12, wherein the poxvirus is selected from
the group consisting of vaccinia, monkeypox, cowpox, rabbitpox,
raccoon pox, tatera pox, buffalopox, and camelpox.
15. The method of claim 12, wherein the poxvirus is selected from
the group consisting of fowl pox, canary pox, goat pox, sheep pox,
lumpy skin disease, myxoma, hare fibroma, orf, pseudo-cowpox,
swinepox, molluscum contagiosum, tanapox, yaba, and mousepox.
16. The method of claim 12, wherein the patient is a human.
17. The method of claim 16, wherein the effective antiviral amount
is from about 300 mg per day to about 15,000 mg per day.
18-24. (canceled)
Description
RELATED APPLICATIONS
[0001] This application claims the benefit of priority of U.S.
Provisional Application No. 60/684,811, filed May 25, 2005, the
disclosure of which is hereby expressly incorporated by reference
in its entirety.
FIELD OF THE INVENTION
[0002] A method for the prevention or treatment of poxvirus
infection by administering an effective amount of an antiviral
agent comprising cyclopropanated carbocyclic 2'-deoxynucleoside to
an individual in need thereof is provided.
BACKGROUND OF THE INVENTION
[0003] Poxviruses (members of the Poxviridae family) are the
largest and most complex viruses. They are linear double-stranded
DNA viruses of 130-300 kilobase pair. The 200-400 nm virion is oval
or brick-shaped and can be visualized by light microscopy. The
extracellular virion possesses 2 envelopes, while the intracellular
virus has only 1 envelope. The virion contains a large number of
proteins, at least 10 of which possess enzymatic activity needed
for genomic replication. Virus replication is equally complex.
Infection is initiated by attachment of the virus to one of several
cellular receptors. The virus then can enter the cell by a number
of mechanisms. Unlike other DNA viruses, poxviruses replicate in
the cytoplasm. The virus contains all the elements for genomic
replication, but cellular functions appear necessary for complete
viral maturation.
[0004] Infections due to the poxviruses occur in humans and
animals. The orthopoxviruses include smallpox (variola), monkeypox,
vaccinia, and cowpox viruses. Parapoxviruses include orf virus,
bovine papular stomatitis virus, pseudocowpox virus, and sealpox
virus. Yatapoxviruses include tanapox virus and yabapoxviruses,
which are found primarily in Africa. Molluscipoxviruses include the
human poxvirus, molluscum contagiosum virus. Smallpox and molluscum
are specific to humans. The other viruses cause rare zoonotic
infections in humans.
[0005] Vaccinia virus can infect humans, and has been used for
immunization against smallpox. Its origins are not known but it is
believed to be a genetically-distinct type of pox virus which grows
readily in a variety of hosts. In man, it causes a localized
pustule with scar formation. In immunocompromised persons or
eczematous persons, it sometimes causes a severe generalized
vaccinia infection. Cowpox is acquired by humans usually by milking
cows. It then manifests as ulcerative lesions (sometimes called
"milkers nodules") on the hands of dairy workers. Despite its name,
rodents are the main reservoir of cowpox; it spreads secondarily to
cows and domestic cats. Vaccinia and cowpox are two of only a few
experimental animal infections available for evaluation of
antiviral compounds against orthopoxviruses.
[0006] Smallpox is transmitted by respiratory route from lesions in
the respiratory tract of patients in the early stage of the
disease. During the 12 day incubation period, the virus is
distributed initially to the internal organs and then to the skin.
Variola major caused severe infections with 20-50% mortality,
variola minor with <1% mortality. Management of outbreaks
depended on the isolation of infected individuals and the
vaccination of close contacts. The vaccine was highly effective. If
given during the incubation period, it either prevented or reduced
the severity of clinical symptoms. The origin of the vaccine strain
is not known, it is thought that it may have been horsepox which is
now an extinct disease. On 8 May 1980, the Thirty-third World
Health Assembly declared that smallpox had been eradicated
globally.
[0007] The discontinuation of routine vaccination has rendered
civilian and military populations more susceptible to a disease
that is not only infectious by aerosol, but also infamous for its
devastating morbidity and mortality. Since 1983, there have existed
two WHO-approved and inspected repositories of variola virus: the
CDC in the United States and Vector Laboratories in Russia. Despite
the promise of variola virus' extinction as a biological entity,
the prospect of surreptitious weaponization of smallpox remains
vexing. Smallpox virus, used as a weapon against an unimmunized
population, has the potential to infect tens of thousands of
individuals, kill 30% or more of those infected, and trigger the
vaccination of many times that number of individuals.
[0008] In most individuals, the smallpox vaccine is safe and
effective. However, certain individuals can experience potentially
fatal effects, including eczema vaccinatum, progressive vaccinia,
and postvaccinal encephalitis. In view of the potential adverse
effects associated with the smallpox vaccine, widespread
vaccination of the population may not be an acceptable response to
the threat of smallpox bioterrorism.
[0009] Most studies have focused so far on the efficacy of
cidofovir (CDV) against poxviruses. (See De Clercq, et al., Reviews
in Medical Virology, September 2004, vol. 14, no. 5, pp.
289-300(12)). This compound has demonstrable activity against
cowpox in mice and against monkeypox in monkeys. In the United
States, cidofovir may be used in emergencies as an investigational
new drug to treat significant adverse events following immunization
with the current smallpox vaccine, and in the unlikely event of
smallpox re-emerging. At present, cidofovir is the drug of choice
for therapy of potential smallpox outbreaks and vaccination
complications, and an investigational new drug protocol was
approved recently for use in response to an actual smallpox
outbreak. Although cidofovir is very active against all the
orthopoxviruses, it has major limitations in its usefulness.
Cidofovir is toxic to kidney tubules and is not active orally,
which necessitates intravenous administration. From a practical
standpoint, it is anticipated that dosing will be limited to a
single dose or to no more than two doses in a smallpox outbreak
except for immunocompromised individuals who may require lengthy
antiviral therapy. (See Quenelle, et al. Antimicrobial Agents and
Chemotherapy, October 2003, p. 3275-3280, Vol. 47, No. 10).
[0010] Nucleoside analogs lacking 2'- and 3'-hydroxyl groups
(dideoxynucleosides), as well as those 2'-deoxynucleosides where
the 3'-hydroxyl function has been chemically modified or changed,
can function as chain terminators of DNA synthesis after their
triphosphate metabolites are incorporated into DNA. This is the
basis of the Sanger dideoxynucleotide method for DNA sequencing
(Sanger et al., Proc. Natl. Acad. Sci. USA, 1977). Intense effort
has focused on the design and use of these compounds as inhibitors
of viral replication (Van Roey et al., Ann. N.Y. Acad. Sci.,
616:29, 1990). Although the conformation of the sugar moiety in
these analogs is believed to play a critical role in modulating
biological activity, including the anti-HIV activity mediated by
derivatives such as azidothymidine (AZT) and dideoxyinosine (ddI),
the main problem encountered in correlating a specific type of
sugar conformation with the biological activity of nucleoside
analogs is that the sugar ring is quite flexible and its
conformation in solution can differ markedly from its conformation
in the solid state (Jagannadh et al., Biochem. Biophys. Res.
Commun., 179:386; Plavec et al., Biochem. Biophys. Methods,
25:253). Thus, for nucleosides in general, any structure-activity
analysis which is based solely on the solid-state conformation
would be inaccurate unless it was previously determined that both
solution and solid-state conformations were the same.
[0011] In solution there is a dynamic equilibrium between Northern
(N) and Southern (S) type furanose conformers (Taylor et al.,
Antiviral Chem. Chemother., 1:163-173, 1990) as defined in the
pseudorotational cycle. In this cycle, an absolute Northern
conformation corresponds to a range of P (angle of pseudorotation)
of from 342.degree. to 18.degree.
(.sub.2E.fwdarw..sup.3T.sub.2.fwdarw..sup.3E), whereas an absolute
Southern conformation corresponds to a range of P of from
162.degree. to 198.degree.
(.sup.2E.fwdarw..sup.2T.sub.3.fwdarw..sub.3E). Preference for any
of these specific conformations in solution is determined by the
interplay of interactions resulting from anomeric and gauche
effects (Saenger, in Principles of Nucleic Acid Structure,
Springer-Verlag, New York, pp. 51-104, 1984; Plavec et al., J. Am.
Chem. Soc., 94:8205-8212, 1972). When a nucleoside or nucleotide
binds to its target enzyme, only one form is expected to be present
at the active site. While the energy gap between Northern and
Southern conformations is about 4 kcal/mol, such a disparity can
explain the difference between micromolar and nanomolar binding
affinities.
[0012] The conformations of nucleosides and their analogs can be
described by the geometry of the glycosyl link (syn or anti), the
rotation about the exocyclic C4'-C5' bond and the puckering of the
sugar ring leading to formation of the twist and envelope
conformations. Two types of sugar puckering are generally
energetically preferred, namely the C2'-exo/C3'-endo (N or
Northern) and the C2'-endo/C3'-exo (S or Southern). The terms
"endo" and "exo" refer to displacement of the atom above or below
the plane of the ribose ring, respectively. The torsion angles
.chi. [C2-N1-C1'-O4' (pyrimidines) or C4-N9-C1'-O4' (purines)] and
.gamma. (C3'-C4'-C5'-O5') describe, respectively, the orientations
of the base and the 5'-hydroxyl group relative to the ribose
ring.
[0013] In DNA duplexes, a Southern conformation of the repeating
nucleoside unit confers upon the double helix a B-conformation,
whereas the Northern conformation induces an A-conformation double
helix. The A and B forms of DNA differ in the number of base pairs
per turn, the amount of rotation per base pair, the vertical rise
per base pair and the helical diameter. In addition, in stretches
of DNA containing alternating purines and pyrimidines, a
left-handed helix called Z-DNA may form.
[0014] Altmann et al. demonstrated that substitution of
N-methanocarba-thymidine ((N)-methanocarba-T) for thymidine in
DNA/RNA heteroduplexes increased the thermodynamic stability of the
double helix, as indicated by a positive increase in the T.sub.m,
whereas the Southern conformer induced a small destabilizing effect
(Altmann et al., Tetrahedron Lett., 35:7625-7628, 1994). The
increased thermal stability reported for two different
(N)-methanocarba-T-containing oligodeoxynucleotides (ODNs) versus
conventional ODNs was between 0.8 and 2.1.degree. for a single
modified nucleotide; however, no data was reported for an ODN
containing multiple (N)-methanocarba-Ts in this study. To further
elucidate the stabilizing effect of multiple (N)-methanocarba-Ts in
the context of the DNA/RNA heteroduplex, a test sequence targeted
to the coding region of the SV40 large T-antigen (Wagner, R. W. et
al. 1993, Science 260:1510-13) was subsequently synthesized as the
phosphorothioate 5'-CTTCATTTTTTCTTC-3' (SEQ ID NO: 1), where all
thymidines (T) were replaced by (N)-methanocarba-Ts (Marquez, et
al., 1996. J. Med. Chem. 39:3739-47). The additive increase in
thermodynamic stability of the heteroduplex due to the presence of
multiple (N)-methanocarba-T nucleotides was clearly demonstrated
with the average stabilization per substitution of ca. 1.3.degree.
C. relative to thymidine (Marquez, et al., 1996. J. Med. Chem.
39:3739-47).
[0015] Conformationally (Northern) locked nucleoside analogs are
described in U.S. Pat. No. 5,629,454 and in U.S. Pat. No.
5,869,666.
SUMMARY OF THE INVENTION
[0016] The threat from a smallpox bioterrorist attack is a serious
health problem for the entire nation. Although early vaccination is
a possible recourse, there is no drug available when that window of
opportunity is missed, especially if an intentional spread of
smallpox occurs in a bioterrorist attack. Accordingly, there is a
need for antiviral agents effective against poxviruses, such as
smallpox. The compositions and methods of the preferred embodiments
provide such agents and associated methods of treatment.
[0017] North-methanocarbathymidine (N-MCT), a thymidine analog with
a pseudosugar moiety locked in the northern conformation, which was
previously shown to exert strong antiviral activity against herpes
simplex virus types 1 and 2 (Marquez, et al., 1996. J. Med. Chem.
39:3739-47), has been identified as exhibiting potent activity
against poxviruses, which are DNA viruses. N-MCT effectively blocks
poxvirus synthesis through its triphosphate (TP) metabolite, which
is more efficiently produced in poxvirus-infected cells. N-MCT is
approximately seven times more potent than cidofovir against
vaccinia and cowpox. The higher potency and target specificity of
N-MCT against poxvirus, as well as its high margin of safety, makes
it a highly desirable antiviral agent. In addition, the antiviral
mechanism of N-MCT may be different from that of cidofovir, making
it even more desirable due to the scarcity of the potential
available efficacious anti-pox agents currently under
development.
[0018] Accordingly, in a first aspect, a method of treating a
poxvirus infection in a mammal in need thereof is provided,
comprising the step of administering, in a pharmaceutically
acceptable carrier, to the mammal an effective antiviral amount of
a compound having the formula:
##STR00001##
wherein R.sub.1 is selected from the group consisting of a
substituted or unsubstituted adenine moiety, a substituted or
unsubstituted thymine moiety, a substituted or unsubstituted
cytosine moiety, and a substituted or unsubstituted guanine
moiety.
[0019] In a second aspect, a method of treating a poxvirus
infection in a mammal in need thereof is provided, comprising the
step of administering, in a pharmaceutically acceptable carrier, to
the mammal an effective antiviral amount of a compound having the
formula:
##STR00002##
[0020] wherein R is selected from the group consisting of: [0021]
hydrogen; [0022] X, wherein X is defined as Cl, F, Br, I; [0023]
hydroxyl; [0024] CH.sub.2--R.sup.2; [0025] CH.dbd.CH--R.sup.2;
and
[0026] C.ident.C--R.sup.2; [0027] an optionally substituted alkoxy
moiety selected from the group consisting of: methoxy, ethoxy,
propoxy, butoxy, sec-butoxy, tert-butoxy, and pentoxy, [0028] an
optionally substituted aryloxy moiety selected from the group
consisting of: phenoxy, benzyloxy, and naphthyloxy; [0029] an
optionally substituted aryl moiety selected from the group
consisting of: phenyl and naphthyl, [0030] an optionally
substituted heterocyclic moiety selected from the group consisting
of: oxiranyl, furanyl, thiofuranyl, pyrrolyl, diazolyl, triazolyl,
dithiolyl, oxathiolyl, oxazolyl, thiazolyl, oxadiazolyl,
oxatriazolyl, dioxazolyl, oxathiazolyl, pyranyl, dioxinyl,
pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, piperazinyl,
triazinyl, oxazinyl, oxathiazinyl, oxadiazinyl, benzofuranyl,
benzothiofuranyl, indolyl, pyranopyrrolyl, benzodiazolyl,
benzoxazolyl, benzopyranyl, benzopyridinyl, pyridopyridinyl, and
benzoxazinyl;
[0031] wherein, when R is a substituted moiety, R is substituted
with a moiety selected from the group consisting of: X, methyl,
ethyl, propyl, butyl, sec-butyl, tert-butyl, pentyl, phenyl,
benzyl, naphthyl, hydroxyl, methoxy, ethoxy, propoxy, butoxy,
sec-butoxy, tert-butoxy, pentoxy, phenoxy and benzyloxy; and
[0032] R.sup.2 is defined as being any one of: H, X, methyl, ethyl,
propyl, butyl, sec-butyl, tert-butyl, pentyl, phenyl, benzyl,
naphthyl, hydroxyl, methoxy, ethoxy, propoxy, butoxy, sec-butoxy,
tert-butoxy, pentoxy, phenoxy, benzyloxy, naphthyloxy, oxiranyl,
furanyl, thiofuranyl, pyrrolyl, diazolyl, triazolyl, dithiolyl,
oxathiolyl, oxazolyl, thiazolyl, oxadiazolyl, oxatriazolyl,
dioxazolyl, oxathiazolyl, pyranyl, dioxinyl, pyridinyl,
pyridazinyl, pyrimidinyl, pyrazinyl, piperazinyl, triazinyl,
oxazinyl, oxathiazinyl, oxadiazinyl, benzofuranyl,
benzothiofuranyl, indolyl, pyranopyrrolyl, benzodiazolyl,
benzoxazolyl, benzopyranyl, benzopyridinyl, pyridopyridinyl, or
benzoxazinyl; and
[0033] further wherein R.sup.2 is not H or X, R.sup.2 may be
optionally substituted by one or more of: X, methyl, ethyl, propyl,
butyl, sec-butyl, tert-butyl, pentyl, phenyl, benzyl, naphthyl,
hydroxyl, methoxy, ethoxy, propoxy, butoxy, sec-butoxy,
tert-butoxy, pentoxy, phenoxy or benzyloxy.
[0034] In an embodiment of the second aspect, the mammal is a
human.
[0035] In an embodiment of the second aspect, the compound is of
the formula:
##STR00003##
[0036] In an embodiment of the second aspect, the poxvirus is
smallpox virus.
[0037] In an embodiment of the second aspect, the poxvirus is
selected from the group consisting of vaccinia, monkeypox, cowpox,
rabbitpox, raccoon pox, tatera pox, buffalopox, and camelpox.
[0038] In an embodiment of the second aspect, the poxvirus is
selected from the group consisting of fowl pox, canary pox, goat
pox, sheep pox, lumpy skin disease, myxoma, hare fibroma, orf,
pseudo-cowpox, swinepox, molluscum contagiosum, tanapox, yaba, and
mousepox.
[0039] In an embodiment of the second aspect, the effective
antiviral amount is from about 300 mg per day to about 15,000 mg
per day.
[0040] In an embodiment of the second aspect, the step of
administering is selected from the group consisting of topically
administering, orally administering, intravenously administering,
intramuscularly administering, parenterally administering,
intradermally administering, intraperitoneally administering, and
subcutaneously administering
[0041] In a third aspect, a method of treating a poxvirus infection
in a mammal in need thereof is provided, comprising the step of
administering to the individual an effective antiviral amount of
North-methanocarbathymidine triphosphate.
[0042] In an embodiment of the third aspect, the mammal is a
human.
[0043] In an embodiment of the third aspect, the poxvirus is
smallpox virus.
[0044] In an embodiment of the third aspect, the poxvirus is
selected from the group consisting of vaccinia, monkeypox, cowpox,
rabbitpox, raccoon pox, tatera pox, buffalopox, and camelpox.
[0045] In an embodiment of the third aspect, the poxvirus is
selected from the group consisting of fowl pox, canary pox, goat
pox, sheep pox, lumpy skin disease, myxoma, hare fibroma, orf,
pseudo-cowpox, swinepox, molluscum contagiosum, tanapox, yaba, and
mousepox.
[0046] In a fourth aspect, a pharmaceutical kit is provided
comprising an antiviral agent comprising a compound having the
formula;
##STR00004##
wherein R.sup.1 is selected from the group consisting of a
substituted or unsubstituted adenine moiety, a substituted or
unsubstituted thymine moiety, a substituted or unsubstituted
cytosine moiety, and a substituted or unsubstituted guanine moiety,
and wherein the antiviral agent is in a pharmaceutically acceptable
carrier; and directions for administering the antiviral agent to a
patient in need thereof for treatment of a poxvirus infection.
[0047] In an embodiment of the fourth aspect, the antiviral agent
is of the formula:
##STR00005##
[0048] wherein R is selected from the group consisting of: [0049]
hydrogen; [0050] X, wherein X is defined as Cl, F, Br, I; [0051]
hydroxyl; [0052] CH.sub.2--R.sup.2; [0053] CH.dbd.CH--R.sup.2; and
[0054] C.ident.C--R.sup.2; [0055] an optionally substituted alkoxy
moiety selected from the group consisting of: methoxy, ethoxy,
propoxy, butoxy, sec-butoxy, tert-butoxy, and pentoxy; [0056] an
optionally substituted aryloxy moiety selected from the group
consisting of: phenoxy, benzyloxy, and naphthyloxy; [0057] an
optionally substituted aryl moiety selected from the group
consisting of: phenyl and naphthyl, [0058] an optionally
substituted heterocyclic moiety selected from the group consisting
of: oxiranyl, furanyl, thiofuranyl, pyrrolyl, diazolyl, triazolyl,
dithiolyl, oxathiolyl, oxazolyl, thiazolyl, oxadiazolyl,
oxatriazolyl, dioxazolyl, oxathiazolyl, pyranyl, dioxinyl,
pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, piperazinyl,
triazinyl, oxazinyl, oxathiazinyl, oxadiazinyl, benzofuranyl,
benzothiofuranyl, indolyl, pyranopyrrolyl, benzodiazolyl,
benzoxazolyl, benzopyranyl, benzopyridinyl, pyridopyridinyl, and
benzoxazinyl;
[0059] wherein, when R is a substituted moiety, R is substituted
with a moiety selected from the group consisting of: X, methyl,
ethyl, propyl, butyl, sec-butyl, tert-butyl, pentyl, phenyl,
benzyl, naphthyl, hydroxyl, methoxy, ethoxy, propoxy, butoxy,
sec-butoxy, tert-butoxy, pentoxy, phenoxy and benzyloxy; and
[0060] R.sup.2 is defined as being any one of: H, X, methyl, ethyl,
propyl, butyl, sec-butyl, tert-butyl, pentyl, phenyl, benzyl,
naphthyl, hydroxyl, methoxy, ethoxy, propoxy, butoxy, sec-butoxy,
tert-butoxy, pentoxy, phenoxy, benzyloxy, naphthyloxy, oxiranyl,
furanyl, thiofuranyl, pyrrolyl, diazolyl, triazolyl, dithiolyl,
oxathiolyl, oxazolyl, thiazolyl, oxadiazolyl, oxatriazolyl,
dioxazolyl, oxathiazolyl, pyranyl, dioxinyl, pyridinyl,
pyridazinyl, pyrimidinyl, pyrazinyl, piperazinyl, triazinyl,
oxazinyl, oxathiazinyl, oxadiazinyl, benzofuranyl,
benzothiofuranyl, indolyl, pyranopyrrolyl, benzodiazolyl,
benzoxazolyl, benzopyranyl, benzopyridinyl, pyridopyridinyl, or
benzoxazinyl; and
[0061] further wherein R.sup.2 is not H or X, R.sup.2 may be
optionally substituted by one or more of: X, methyl, ethyl, propyl,
butyl, sec-butyl, tert-butyl, pentyl, phenyl, benzyl, naphthyl,
hydroxyl, methoxy, ethoxy, propoxy, butoxy, sec-butoxy,
tert-butoxy, pentoxy, phenoxy or benzyloxy.
[0062] In an embodiment of the fourth aspect, the antiviral agent
is of the formula:
##STR00006##
[0063] In an embodiment of the fourth aspect, the patient is a
human.
[0064] In an embodiment of the fourth aspect, the poxvirus is
smallpox virus.
[0065] In an embodiment of the fourth aspect, the poxvirus is
selected from the group consisting of vaccinia, monkeypox, cowpox,
rabbitpox, raccoon pox, tatera pox, buffalopox, and camelpox.
[0066] In an embodiment of the fourth aspect, the poxvirus is
selected from the group consisting of fowl pox, canary pox, goat
pox, sheep pox, lumpy skin disease, myxoma, hare fibroma, orf,
pseudo-cowpox, swinepox, molluscum contagiosum, tanapox, yaba, and
mousepox.
[0067] In an embodiment of the fourth aspect, the kit further
comprises cidofovir and directions for administering the cidofovir
to the patient.
[0068] In an embodiment of the fourth aspect, the kit further
comprises an anti-infective agent and directions for administering
the anti-infective agent to the patient in need thereof for
treatment of a secondary skin infection associated with smallpox
virus infection.
BRIEF DESCRIPTION OF THE DRAWINGS
[0069] FIG. 1 depicts multiple amino acid alignments of thymidine
kinase homologs expressed in herpes simplex virus 1 (HSV-1), cowpox
virus (CV), vaccinia virus (VV) and humans, including a consensus
line. Identity and similarity are depicted as light gray and dark
gray boxes, respectively.
[0070] FIG. 2 depicts an unrooted phylogenetic tree based on the
amino acid sequence of the thymidine kinase (TK) homologs is shown.
Type II orthopoxvirus TK homologs are closely related to the human
enzyme that is a member of this same class.
[0071] FIG. 3 depicts inhibition of VV DNA synthesis by N-MCT.
Monolayers of HFF cells were infected with the WR strain of VV and
incubated with increasing concentrations of the drugs as
illustrated in the figure (0.03, 0.1, 3, 10, and 30 .mu.g/ml).
Purified DNA was cut with EcoRV, and a 3,259-bp fragment of VV DNA
was detected with a digoxigenin labeled DNA probe. Numbers at left
are molecular sizes in base pairs. CC and VC designate cell control
and virus control, respectively.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
[0072] The following description and examples illustrate some
exemplary embodiments of the disclosed invention in detail. Those
of skill in the art will recognize that there are numerous
variations and modifications of this invention that are encompassed
by its scope. Accordingly, the description of a certain exemplary
embodiment should not be deemed to limit the scope of the present
invention.
[0073] The carbocyclic 2'-deoxynucleoside analogs of preferred
embodiments can be employed in the treatment and prevention of
infection by poxviruses. The poxvirus family is subdivided into the
entomopoxvirus (EnPV) and chordopoxvirus (ChPV) subfamilies
(Entomopoxvirinae and Chordopoxvirinae), which infect insects and
chordates, respectively. The ChPVs are further divided into eight
genera (Avipoxvirus, Molluscipoxvirus, Orthopoxvirus,
Capripoxvirus, Suipoxvirus, Leporipoxvirus, Yatapoxvirus and
Parapoxvirus), whereas the EnPVs are divided into three genera (A,
B and C). The genus Orthopoxvirus includes smallpox (variola),
vaccinia, monkeypox, cowpox, rabbitpox, raccoon pox, tatera pox,
buffalopox, and camelpox. The genus Avipoxvirus includes fowlpox
and canary pox. The genus Capripoxvirus includes goat pox, sheep
pox, and lumpy skin disease. The genus Leporipoxvirus includes
myxoma and hare fibroma. The genus Parapoxvirus includes orf and
pseudo-cowpox. The genus Suipoxvirus includes swinepox. The genus
Molluscipoxvirus includes molluscum contagiosum. The genus
Yatapoxvirus includes tanapox and yaba. Other members of the
Poxviridae family include Ectromelia (mousepox) virus. While the
preferred embodiments are generally described in relation to
smallpox, it is understood that the formulations and methods of
preferred embodiments are suitable for use in connection with the
specific poxviruses referred to herein as well as other poxviruses.
Natural poxviruses, as well as genetically engineered or modified
poxviruses and poxviruses resistant to conventional therapies, are
also amenable for treatment or prevention by the formulations and
methods of preferred embodiments.
Cyclopropanated Carbocyclic 2'-Deoxynucleosides
[0074] Carbocyclic 2'-deoxynucleoside analogs locked in the
Northern conformation are effective agents in the prevention and
treatment of poxvirus infections. These compounds are described in
U.S. Pat. No. 5,629,454 and in U.S. Pat. No. 5,869,666.
Conformationally rigid (locked) nucleoside analogs are constructed
on a bicyclo[3.1.0]hexane template whose value of P
(pseudorotational angle) fits within the range of absolute Northern
or Southern conformations. This bicyclo[3.1.0]hexane template
exists exclusively as a pseudoboat, and carbocyclic nucleosides
built thereon can adopt either a Northern or Southern conformation,
depending on the relative disposition of substituents on the ring.
Thus, a Northern C2'-exo (2E) envelope conformation is obtained
when the cyclopropane ring was fused between carbon C4' and the
carbon supplanting the ribofuranoside oxygen. Conversely, fusion of
the cyclopropane ring between carbon C1' and the carbon supplanting
the ribofuranoside oxygen provides the opposite Southern
conformation. The cyclopropanated carbocyclic 2'-deoxynucleosides
of preferred embodiments have the formula:
##STR00007##
wherein R.sub.1 is adenine, an adenine derivative, a substituted
adenine, guanine, a guanine derivative, a substituted guanine,
cytosine, a cytosine derivative, a substituted cytosine, thymine, a
thymine derivative, a substituted thymine, uracil, a uracil
derivative, or a substituted uracil; R.sub.2 and R.sub.3 are
independently selected from hydrogen, alkyl, alkylaryl, aryl,
arylalkyl, alkoxy, alkyloxyalkyl, alkyloxyaryl, aryloxyalkyl,
alkylaryloxy, aryloxy, and arylalkyloxy. If R.sub.2 or R.sub.3 is a
moiety other than hydrogen, then it can be substituted, for
example, by one or more halogen atoms. In a preferred embodiment,
the compounds exhibit the following stereochemistry:
##STR00008##
Particularly preferred are compounds of such stereochemistry
wherein R.sub.2 is hydroxyl, R.sub.3 is hydrogen, and R.sub.1 is
selected from the group consisting of adenine, thymine, cytosine,
and guanine. While this particular stereochemistry is generally
preferred in many embodiments, other stereochemistries can also be
preferred in certain other embodiments.
[0075] The thymine, uracil, and substituted uracil cyclopropanated
carbocyclic 2'-deoxynucleosides are particularly preferred
compounds. These compounds have the structure:
##STR00009##
[0076] wherein R is selected from the group consisting of: [0077]
hydrogen; [0078] X, wherein X is defined as Cl, F, Br, I; [0079]
hydroxyl; [0080] CH.sub.2--R.sup.2; [0081] CH.dbd.CH--R.sup.2;
and
[0082] C.ident.C--R.sup.2; [0083] an optionally substituted alkoxy
moiety selected from the group consisting of: methoxy, ethoxy,
propoxy, butoxy, sec-butoxy, tert-butoxy, and pentoxy; [0084] an
optionally substituted aryloxy moiety selected from the group
consisting of: phenoxy, benzyloxy, and naphthyloxy; [0085] an
optionally substituted aryl moiety selected from the group
consisting of: phenyl and naphthyl, [0086] an optionally
substituted heterocyclic moiety selected from the group consisting
of: oxiranyl, furanyl, thiofuranyl, pyrrolyl, diazolyl, triazolyl,
dithiolyl, oxathiolyl, oxazolyl, thiazolyl, oxadiazolyl,
oxatriazolyl, dioxazolyl, oxathiazolyl, pyranyl, dioxinyl,
pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, piperazinyl,
triazinyl, oxazinyl, oxathiazinyl, oxadiazinyl, benzofuranyl,
benzothiofuranyl, indolyl, pyranopyrrolyl, benzodiazolyl,
benzoxazolyl, benzopyranyl, benzopyridinyl, pyridopyridinyl, and
benzoxazinyl;
[0087] wherein, when R is a substituted moiety, R is substituted
with a moiety selected from the group consisting of: X, methyl,
ethyl, propyl, butyl, sec-butyl, tert-butyl, pentyl, phenyl,
benzyl, naphthyl, hydroxyl, methoxy, ethoxy, propoxy, butoxy,
sec-butoxy, tert-butoxy, pentoxy, phenoxy and benzyloxy; and
[0088] R.sup.2 is defined as being any one of: H, X, methyl, ethyl,
propyl, butyl, sec-butyl, tert-butyl, pentyl, phenyl, benzyl,
naphthyl, hydroxyl, methoxy, ethoxy, propoxy, butoxy, sec-butoxy,
tert-butoxy, pentoxy, phenoxy, benzyloxy, naphthyloxy, oxiranyl,
furanyl, thiofuranyl, pyrrolyl, diazolyl, triazolyl, dithiolyl,
oxathiolyl, oxazolyl, thiazolyl, oxadiazolyl, oxatriazolyl,
dioxazolyl, oxathiazolyl, pyranyl, dioxinyl, pyridinyl,
pyridazinyl, pyrimidinyl, pyrazinyl, piperazinyl, triazinyl,
oxazinyl, oxathiazinyl, oxadiazinyl, benzofuranyl,
benzothiofuranyl, indolyl, pyranopyrrolyl, benzodiazolyl,
benzoxazolyl, benzopyranyl, benzopyridinyl, pyridopyridinyl, or
benzoxazinyl; and
[0089] further wherein R.sup.2 is not H or X, R.sup.2 may be
optionally substituted by one or more of: X, methyl, ethyl, propyl,
butyl, sec-butyl, tert-butyl, pentyl, phenyl, benzyl, naphthyl,
hydroxyl, methoxy, ethoxy, propoxy, butoxy, sec-butoxy,
tert-butoxy, pentoxy, phenoxy or benzyloxy.
[0090] Thymine and substituted uracil cyclopropanated carbocyclic
2'-deoxynucleosides are particularly preferred:
##STR00010##
[0091] The term "alkyl," as used herein is a broad term and is used
in its ordinary sense, including, without limitation, to refer to a
straight chain or branched, acyclic or cyclic, unsaturated or
saturated aliphatic hydrocarbon containing 1, 2, 3, 4, 5, 6, 7, 8,
9, or 10 or more carbon atoms, while the term "lower alkyl" has the
same meaning as alkyl but contains 1, 2, 3, 4, 5, or 6 carbon
atoms. Representative saturated straight chain alkyls include
methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, and the like;
while saturated branched alkyls include isopropyl, sec-butyl,
isobutyl, ter-t-butyl, isopentyl, and the like. Unsaturated alkyls
contain at least one double or triple bond between adjacent carbon
atoms (referred to as an "alkenyl" or "alkynyl," respectively).
Representative straight chain and branched alkenyls include
ethylenyl, propylenyl, 1-butenyl, 2-butenyl, isobutylenyl,
1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl,
2,3-dimethyl-2-butenyl, and the like; while representative straight
chain and branched alkynyls include acetylenyl, propynyl,
1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3-methyl-1 butynyl,
and the like. The term "cycloalkyl," as used herein is a broad term
and is used in its ordinary sense, including, without limitation,
to refer to alkyls that include mono-, di-, or poly-homocyclic
rings. Cycloalkyls are also referred to as "cyclic alkyls" or
"homocyclic rings." Representative saturated cyclic alkyls include
cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,
--CH.sub.2cyclopropyl, --CH.sub.2cyclobutyl, --CH.sub.2cyclopentyl,
--CH.sub.2cyclohexyl, and the like; while unsaturated cyclic alkyls
include cyclopentenyl and cyclohexenyl, and the like. Cyclic alkyls
include decalin, adamantane, and the like.
[0092] The term "aryl," as used herein is a broad term and is used
in its ordinary sense, including, without limitation, to refer to
an aromatic carbocyclic moiety such as phenyl or naphthyl. The term
"arylalkyl," as used herein is a broad term and is used in its
ordinary sense, including, without limitation, to refer to an alkyl
having at least one alkyl hydrogen atom replaced with an aryl
moiety, such as benzyl, --CH.sub.2(1-naphthyl),
--CH.sub.2(2-naphthyl), --(CH.sub.2).sub.2-phenyl,
--(CH.sub.2).sub.3-phenyl, --CH(phenyl).sub.2, and the like.
[0093] The term "substituted," as used herein is a broad term and
is used in its ordinary sense, including, without limitation, to
refer to any of the above groups wherein at least one hydrogen atom
is replaced with a substituent. In the case of a keto substituent
(i.e., --C(.dbd.O)--) two hydrogen atoms are replaced. When
substituted, "substituents," within the context of preferred
embodiments, include halogen, hydroxy, cyano, nitro, amino,
alkylamino, dialkylamino, alkyl, alkoxy, alkylthio, haloalkyl,
aryl, substituted aryl, arylalkyl, substituted arylalkyl,
heteroaryl, substituted heteroaryl, heteroarylalkyl, substituted
heteroarylalkyl, heterocycle, substituted heterocycle,
heterocyclealkyl, substituted heterocyclealkyl, --NR.sub.aR.sub.b,
--NR.sub.aC(.dbd.O)R.sub.b, --NR.sub.aC(.dbd.O)NR.sub.bR.sub.c,
--NR.sub.aC(.dbd.O)OR.sub.b, --NR.sub.aSO.sub.2R.sub.b, --OR.sub.a,
--C(.dbd.O)R.sub.a, --C(.dbd.O)OR.sub.a,
--C(.dbd.O)NR.sub.aR.sub.b, --SH, --SR.sub.0, --SOR.sub.a,
--S(.dbd.O).sub.2R.sub.a, --OS(.dbd.O).sub.2R.sub.a,
--OC(.dbd.O)NR.sub.aR.sub.b, --S(.dbd.O).sub.2OR.sub.a, wherein
R.sub.a, R.sub.b, and R.sub.c are the same or different and are
independently selected from hydrogen, alkyl, haloalkyl, substituted
alkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl,
heteroaryl, substituted heteroaryl, heteroarylalkyl, substituted
heteroarylalkyl, heterocycle, substituted heterocycle,
heterocyclealkyl or substituted heterocyclealkyl. When a ring
system (e.g., a fused ring system, a heterocyclic ring system, a
homocyclic ring system, and/or any other ring system) is
substituted, the substituents can occupy any location on the ring
or a chain attached to the ring.
[0094] The term "halogen," as used herein is a broad term and is
used in its ordinary sense, including, without limitation, to refer
to fluoro, chloro, bromo, and iodo. The term "haloalkyl," as used
herein is a broad term and is used in its ordinary sense,
including, without limitation, to refer to an alkyl having at least
one hydrogen atom replaced with halogen, such as trifluoromethyl
and the like. The term "alkoxy," as used herein is a broad term and
is used in its ordinary sense, including, without limitation, to
refer to an alkyl moiety attached through an oxygen bridge (i.e.,
--O-alkyl) such as methoxy, ethoxy, and the like. The term
"hydroxyalkyl" as used herein is a broad term and is used in its
ordinary sense, including, without limitation, to refer to an alkyl
substituted with at least one hydroxyl group. The term "mono- or
di-(cycloalkyl)methyl," as used herein is a broad term and is used
in its ordinary sense, including, without limitation, to refer to a
methyl group substituted with one or two cycloalkyl groups, such as
cyclopropylmethyl, dicyclopropylmethyl, and the like.
[0095] The term "alkyloxyalkyl" as used herein is a broad term and
is used in its ordinary sense, including, without limitation, to
refer to an alkyl substituted with an &alkyl group. The cyclic
systems referred to herein include fused ring, bridged ring, and
spiro ring moieties, in addition to isolated monocyclic
moieties.
[0096] The compounds of preferred embodiments can incorporate
substituents comprising various ring systems. For each ring system,
any ring atom capable of being an attachment point can comprise a
"point of attachment" of the ring system to the rest of the
molecule. Accordingly, in heterocyclic ring systems, any
hetero-ring atom can be the point of attachment or occupy another
location relative to the point of attachment. In fused ring
systems, there can be multiple points of attachments. For rings
containing more than one hetero-ring atom, the ring name is
intended to cover multiple locations for the hetero-atoms in the
ring. For example, the term "triazolyl" encompasses both
1,2,3-triazolyl and 1,2,4-triazole; the term "diazolyl" encompasses
1,2-diazolyl (i.e., pyrazolyl) and 1,3-diazolyl (i.e., imidazolyl).
Other ring systems wherein the ring name encompasses multiple
locations for the heteroatom(s) in the ring include dithiolyl,
oxathiolyl, oxazolyl, thiazolyl, oxadiazolyl, oxatriazolyl,
dioxazolyl, oxathiazolyl, dioxinyl, triazinyl, oxazinyl,
oxathiazinyl, and oxadiazinyl ring systems. Moreover, for fused
ring systems wherein at least one ring comprises at least one
heteroatom, the heteroatom can occupy any location on the
respective ring. For example the term "benzopyridinyl" encompasses
benzo[b]pyridinyl (i.e., quinolinyl) as well as benzo[c]pyridinyl
(i.e., isoquinolinyl). Other fused ring systems wherein the ring
name encompasses multiple locations for the heteroatom(s) in the
ring include benzofuranyl, benzothiofuranyl, indolyl,
pyranopyrrolyl, benzodiazolyl, benzoxazolyl, benzopyranyl,
pyridopyridinyl, and benzoxazinyl fused ring systems.
[0097] Preferred cyclopropanated carbocyclic 2'-deoxynucleosides
include (N)-2'-deoxy-methanocarba-A (adenosine analog),
(N)-methanocarba-T (thymidine analog), (N)-2'-deoxy-methanocarba-G
(guanosine analog), (N)-2'-deoxy-methanocarba-C (cytosine analog)
and (N)-2'-deoxy-methanocarba-U (uridine analog). These particular
cyclopropanated carbocyclic 2'-deoxynucleosides are depicted by the
following structure:
##STR00011##
wherein B is adenine, thymine, cytosine, guanine or uracil.
[0098] The thymine analogs, especially North-methanocarbathymidine
(N-MCT), are particularly preferred because of their exceptionally
potent activity against poxviruses such as cowpox and vaccinia.
##STR00012##
[0099] The synthesis of the (N)-methanocarbathymidine (or its
adenosine, guanine, cytidine, or uridine analogs) is described
below and in Schemes 1-4. The anti-pox effect of various
substituted derivatives of the (N)-methanocarba 2'-deoxynucleoside
analogs described below can easily be determined by one of ordinary
skill in the art using the assay methods described herein without
undue experimentation.
[0100] Schemes 1-2 can be utilized for the synthesis of
intermediate 12, which is chiral, so there is no need for optical
resolution at the end of the synthesis, and which can be employed
as a starting material for the synthesis of related carbocyclic
2'-deoxynucleoside analogs. Cyclopentenol 6 can be obtained from
the sodium borohydride reduction of cyclopentenone 5 (Marquez et
al., J. Org. Chem., 53:5709, 1988). Regioselective cleavage of the
contiguous O-isopropylidenetriol system in 6 with trimethylaluminum
(Takano et al., Tetrahedron Lett., 29:1823, 1988) produces the
corresponding carbocyclic 3-tert-butoxy-1,5-glycol 7, which in the
presence of tert-butyldimethylsilyl chloride reacts exclusively at
the less hindered allylic alcohol position to give the protected
intermediate 8. Barton's radical deoxygenation of 8 at C-5 occurs
via the xanthate 9 in the presence of AIBN to give compound 10.
Deprotection of the silyl ether in 10 by fluoride ion unmasks the
hydroxyl group (compound 11) which directs the ensuing
cyclopropanation to give compound 12.
##STR00013##
[0101] This compound is directly coupled to 6-chloropurine under
Mitsunobu conditions (Mitsunobu, Synthesis, 1:1-28, 1981) to give
the protected carbocyclic nucleoside intermediate 13. Following
aminolysis of 13 with ammonia, and the simultaneous removal of both
benzyl and tert-butyl groups, the (N)-2'-deoxy-methanocarba
adenosine derivative 4 is obtained.
##STR00014##
[0102] For the pyrimidine derivatives (Scheme 3), protected
N.sup.3-benzoylthymine and N.sup.3-benzoyluracil (Cruickshank et
al., Tetrahedron Lett., 25:681, 1994) are coupled according to
Scheme 3. In the case of 16, the O-alkylated product predominates,
whereas for the uracil analog 17, the situation is reversed.
Base-catalyzed deprotection of the N-benzoyl group from
intermediates 16 and 17 yields the penultimate intermediates 18 and
19, respectively, and simultaneous removal of both O-benzyl and
O-tert-butyl groups with BCl.sub.3 provide the desired targets
(N)-methanocarba-T 20 and (N)-methanocarba-U 21. (N)-methanocarba-C
22 is prepared from (N)-methanocarba-U 21 via formation of the
triazole intermediate (Divakar et al., J. Chem. Soc. Perkin.
Trans., 1, 1171-1176, 1982).
##STR00015##
[0103] For the synthesis of (N)-methanocarba-G 24 (Scheme 4),
coupling under Mitsunobu conditions proceeds with a yield
comparable to that of the pyrimidines. Only the desired N-9 isomer
(34%) is obtained with virtually no detection of the N-7 isomer.
The conversion of the 2-amino-6-chloro intermediate into the
6-O-benzyl derivative 23 facilitates the one-step removal of all
protective groups in the generation of the guanine base (Rodriguez
et al., Tetrahedron Lett., 34:6233-6236, 1993; Rodriguez et al., J.
Med. Chem., 37:3389-3399, 1994).
##STR00016##
[0104] The cyclopropanated carbocyclic 2'-deoxynucleosides of
preferred embodiments can also be incorporated into short
oligodeoxynucleotides (ODNs). Standard double helices exist in the
classic B-DNA form, in which all sugars have a Southern
conformation, or in the A-DNA form, wherein the sugars have an
N-conformation. During formation of DNA/RNA heteroduplexes, the
A-form, typical of RNA, is dominant. The expected thermodynamic
stability resulting from the preorganization of the pseudosugar
rings into the Northern conformation, typical of A-DNA, is evident
by the increase in melting temperature (T.sub.m) of the
corresponding DNA/RNA heteroduplex containing the (N)-methanocarba
T.
Antiviral Compositions Comprising Cyclopropanated Carbocyclic
2'-Deoxynucleosides
[0105] The cyclopropanated carbocyclic 2'-deoxynucleosides (or
derivatives, nucleoside prodrugs, or pharmaceutically acceptable
esters or salts thereof) of the preferred embodiments, can be
incorporated into a pharmaceutically acceptable carrier for
administration to an individual having a poxvirus infection, such
as a smallpox infection, or can be administered prophylactically to
prevent infection upon exposure to a poxvirus. The cyclopropanated
carbocyclic 2'-deoxynucleoside can be employed as the sole agent in
the prevention or treatment of poxvirus infection, or two or more
cyclopropanated carbocyclic 2'-deoxynucleosides can be employed,
optionally in combination with other therapeutic agents, e.g.,
other drugs employed in the treatment of poxvirus infection or in
the alleviation of symptoms of poxvirus infection.
[0106] The terms "pharmaceutically acceptable salts" and "a
pharmaceutically acceptable salt thereof" as used herein are broad
terms and are used in their ordinary sense, including, without
limitation, to refer to salts prepared from pharmaceutically
acceptable, non-toxic acids or bases. Suitable pharmaceutically
acceptable salts include metallic salts, e.g., salts of aluminum,
zinc, alkali metal salts such as lithium, sodium, and potassium
salts, alkaline earth metal salts such as calcium and magnesium
salts; organic salts, e.g., salts of lysine,
N,N'-dibenzylethylenediamine, chloroprocaine, choline,
diethanolamine, ethylenediamine, meglumine (N-methylglucamine),
procaine, and tris; salts of free acids and bases; inorganic salts,
e.g., sulfate, hydrochloride, and hydrobromide; and other salts
which are currently in widespread pharmaceutical use and are listed
in sources well known to those of skill in the art, such as, for
example, The Merck Index. Any suitable constituent can be selected
to make a salt of the cyclopropanated carbocyclic
2'-deoxynucleoside or other therapeutic agents discussed herein,
provided that it is non-toxic and does not substantially interfere
with the desired activity. In addition to salts, pharmaceutically
acceptable precursors and derivatives of the compounds can be
employed. Pharmaceutically acceptable amides, lower alkyl esters,
and protected derivatives can also be suitable for use in
compositions and methods of preferred embodiments.
[0107] Contemplated routes of administration include topical, oral,
intravenous, subcutaneous, parenteral, intradermal, intramuscular,
intraperitoneal, and intravenous, including injectable
administration, sustained release from implants, administration by
eyedrops, and the like. Nonlimiting examples of particularly
preferred nucleoside analog compositions for topical administration
include creams, lotions, gels, salves, sprays, dispersions,
suspensions, pastes, and ointments.
[0108] The cyclopropanated carbocyclic 2'-deoxynucleosides of
preferred embodiments can be formulated into liquid preparations
for, e.g., oral, nasal, anal, rectal, buccal, vaginal, peroral,
intragastric, mucosal, perlingual, alveolar, gingival, olfactory,
or respiratory mucosa administration. Suitable forms for such
administration include suspensions, syrups, and elixirs. If nasal
or respiratory (mucosal) administration is desired (e.g., aerosol
inhalation or insufflation), compositions may be in a form and
dispensed by a squeeze spray dispenser, pump dispenser or aerosol
dispenser. Aerosols are usually under pressure by means of a
hydrocarbon. Pump dispensers can preferably dispense a metered dose
or a dose having a particular particle size.
[0109] The pharmaceutical compositions containing cyclopropanated
carbocyclic 2'-deoxynucleosides are preferably isotonic with the
blood or other body fluid of the recipient. The isotonicity of the
compositions can be attained using sodium tartrate, propylene
glycol or other inorganic or organic solutes. Sodium chloride is
particularly preferred. Buffering agents can be employed, such as
acetic acid and salts, citric acid and salts, boric acid and salts,
and phosphoric acid and salts. Parenteral vehicles include sodium
chloride solution, Ringer's dextrose, dextrose and sodium chloride,
lactated Ringer's or fixed oils. Intravenous vehicles include fluid
and nutrient replenishers, electrolyte replenishers (such as those
based on Ringer's dextrose), and the like.
[0110] Viscosity of the pharmaceutical compositions can be
maintained at the selected level using a pharmaceutically
acceptable thickening agent. Methylcellulose is preferred because
it is readily and economically available and is easy to work with.
Other suitable thickening agents include, for example, xanthan gum,
carboxymethyl cellulose, hydroxypropyl cellulose, carbomer, and the
like. The preferred concentration of the thickener will depend upon
the thickening agent selected. An amount is preferably used that
will achieve the selected viscosity. Viscous compositions are
normally prepared from solutions by the addition of such thickening
agents.
[0111] A pharmaceutically acceptable preservative can be employed
to increase the shelf life of the pharmaceutical compositions.
Benzyl alcohol can be suitable, although a variety of preservatives
including, for example, parabens, thimerosal, chlorobutanol, or
benzalkonium chloride can also be employed. A suitable
concentration of the preservative is typically from about 0.02% to
about 2% based on the total weight of the composition, although
larger or smaller amounts can be desirable depending upon the agent
selected.
[0112] The cyclopropanated carbocyclic 2'-deoxynucleosides of
preferred embodiments can be in admixture with a suitable carrier,
diluent, or excipient such as sterile water, physiological saline,
glucose, or the like, and can contain auxiliary substances such as
wetting or emulsifying agents, pH buffering agents, gelling or
viscosity enhancing additives, preservatives, flavoring agents,
colors, and the like, depending upon the route of administration
and the preparation desired. Standard texts, such as "Remington:
The Science and Practice of Pharmacy", Lippincott Williams &
Wilkins; 20th edition (Jun. 1, 2003) and "Remington's
Pharmaceutical Sciences," Mack Pub. Co.; 18.sup.th and 19.sup.th
editions (December 1985, and June 1990, respectively). Such
preparations can include complexing agents, metal ions, polymeric
compounds such as polyacetic acid, polyglycolic acid, hydrogels,
dextran, and the like, liposomes, microemulsions, micelles,
unilamellar or multilamellar vesicles, erythrocyte ghosts or
spheroblasts. Suitable lipids for liposomal formulation include,
without limitation, monoglycerides, diglycerides, sulfatides,
lysolecithin, phospholipids, saponin, bile acids, and the like. The
presence of such additional components can influence the physical
state, solubility, stability, rate of in vivo release, and rate of
in vivo clearance, and are thus chosen according to the intended
application, such that the characteristics of the carrier are
tailored to the selected route of administration.
[0113] For oral administration, the cyclopropanated carbocyclic
2'-deoxynucleosides can be provided as a tablet, aqueous or oil
suspension, dispersible powder or granule, emulsion, hard or soft
capsule, syrup or elixir. Compositions intended for oral use can be
prepared according to any method known in the art for the
manufacture of pharmaceutical compositions and can include one or
more of the following agents: sweeteners, flavoring agents,
coloring agents and preservatives. Aqueous suspensions can contain
the active ingredient in admixture with excipients suitable for the
manufacture of aqueous suspensions.
[0114] Formulations for oral use can also be provided as hard
gelatin capsules, wherein the cyclopropanated carbocyclic
2'-deoxynucleoside is mixed with an inert solid diluent, such as
calcium carbonate, calcium phosphate, or kaolin, or as soft gelatin
capsules. In soft capsules, the active compounds can be dissolved
or suspended in suitable liquids, such as water or an oil medium,
such as peanut oil, olive oil, fatty oils, liquid paraffin, or
liquid polyethylene glycols. Stabilizers and microspheres
formulated for oral administration can also be used. Capsules can
include push-fit capsules made of gelatin, as well as soft, sealed
capsules made of gelatin and a plasticizer, such as glycerol or
sorbitol. The push-fit capsules can contain the cyclopropanated
carbocyclic 2'-deoxynucleoside in admixture with fillers such as
lactose, binders such as starches, and/or lubricants such as talc
or magnesium stearate and, optionally, stabilizers.
[0115] Tablets can be uncoated or coated by known methods to delay
disintegration and absorption in the gastrointestinal tract and
thereby provide a sustained action over a longer period of time.
For example, a time delay material such as glyceryl monostearate
can be used. When administered in solid form, such as tablet form,
the solid form typically comprises from about 0.001 wt. % or less
to about 50 wt. % or more of active ingredient(s) including the
cyclopropanated carbocyclic 2'-deoxynucleoside, preferably from
about 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09,
0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 wt. % to about 2,
3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, or 45 wt. %.
[0116] Tablets can contain the cyclopropanated carbocyclic
2'-deoxynucleoside in admixture with non-toxic pharmaceutically
acceptable excipients including inert materials. For example, a
tablet can be prepared by compression or molding, optionally, with
one or more additional ingredients. Compressed tablets can be
prepared by compressing in a suitable machine the active
ingredients in a free-flowing form such as powder or granules,
optionally mixed with a binder, lubricant, inert diluent, surface
active or dispersing agent. Molded tablets can be made by molding,
in a suitable machine, a mixture of the powdered compound moistened
with an inert liquid diluent.
[0117] Preferably, each tablet or capsule contains from about 10 mg
or less to about 1,000 mg or more of the cyclopropanated
carbocyclic 2'-deoxynucleoside, more preferably from about 20, 30,
40, 50, 60, 70, 80, 90, or 100 mg to about 150, 200, 250, 300, 350,
400, 450, 500, 550, 600, 650, 700, 750, 800, or 900 mg. Most
preferably, tablets or capsules are provided in a range of dosages
to permit divided dosages to be administered. A dosage appropriate
to the patient and the number of doses to be administered daily can
thus be conveniently selected. While it is generally preferred to
incorporate the cyclopropanated carbocyclic 2'-deoxynucleoside and
any other therapeutic agent employed in combination therewith in a
single tablet or other dosage form, e.g., in a combination therapy,
in certain embodiments it can be desirable to provide the
cyclopropanated carbocyclic 2'-deoxynucleoside and other
therapeutic agents in separate dosage forms.
[0118] Suitable inert materials include diluents, such as
carbohydrates, mannitol, lactose, anhydrous lactose, cellulose,
sucrose, modified dextrans, starch, and the like, or inorganic
salts such as calcium triphosphate, calcium phosphate, sodium
phosphate, calcium carbonate, sodium carbonate, magnesium
carbonate, and sodium chloride. Disintegrants or granulating agents
can be included in the formulation, for example, starches such as
corn starch, alginic acid, sodium starch glycolate, Amberlite,
sodium carboxymethylcellulose, ultramylopectin, sodium alginate,
gelatin, orange peel, acid carboxymethyl cellulose, natural sponge
and bentonite, insoluble cationic exchange resins, powdered gums
such as agar, karaya or tragacanth, or alginic acid or salts
thereof.
[0119] Binders can be used to form a hard tablet. Binders include
materials from natural products such as acacia, tragacanth, starch
and gelatin, methyl cellulose, ethyl cellulose, carboxymethyl
cellulose, polyvinyl pyrrolidone, hydroxypropylmethyl cellulose,
and the like.
[0120] Lubricants, such as stearic acid or magnesium or calcium
salts thereof, polytetrafluoroethylene, liquid paraffin, vegetable
oils and waxes, sodium lauryl sulfate, magnesium lauryl sulfate,
polyethylene glycol, starch, talc, pyrogenic silica, hydrated
silicoaluminate, and the like, can be included in tablet
formulations.
[0121] Surfactants can also be employed, for example, anionic
detergents such as sodium lauryl sulfate, dioctyl sodium
sulfosuccinate and dioctyl sodium sulfonate, cationic such as
benzalkonium chloride or benzethonium chloride, or nonionic
detergents such as polyoxyethylene hydrogenated castor oil,
glycerol monostearate, polysorbates, sucrose fatty acid ester,
methyl cellulose, or carboxymethyl cellulose.
[0122] Controlled release formulations can be employed wherein the
cyclopropanated carbocyclic 2'-deoxynucleoside is incorporated into
an inert matrix that permits release by either diffusion or
leaching mechanisms. Slowly degenerating matrices can also be
incorporated into the formulation. Other delivery systems can
include timed release, delayed release, or sustained release
delivery systems.
[0123] Coatings can be used, for example, nonenteric materials such
as methyl cellulose, ethyl cellulose, hydroxyethyl cellulose,
methylhydroxy-ethyl cellulose, hydroxypropyl cellulose,
hydroxypropyl-methyl cellulose, sodium carboxy-methyl cellulose,
providone and the polyethylene glycols, or enteric materials such
as phthalic acid esters. Dyestuffs or pigments can be added for
identification or to characterize different combinations of active
compound doses
[0124] When administered orally in liquid form, a liquid carrier
such as water, petroleum, oils of animal or plant origin such as
peanut oil, mineral oil, soybean oil, or sesame oil, or synthetic
oils can be added to the cyclopropanated carbocyclic
2'-deoxynucleoside. Physiological saline solution, dextrose, or
other saccharide solution, or glycols such as ethylene glycol,
propylene glycol, or polyethylene glycol are also suitable liquid
carriers. The pharmaceutical compositions can also be in the form
of oil-in-water emulsions. The oily phase can be a vegetable oil,
such as olive or arachis oil, a mineral oil such as liquid
paraffin, or a mixture thereof. Suitable emulsifying agents include
naturally-occurring gums such as gum acacia and gum tragacanth,
naturally occurring phosphatides, such as soybean lecithin, esters
or partial esters derived from fatty acids and hexitol anhydrides,
such as sorbitan mono-oleate, and condensation products of these
partial esters with ethylene oxide, such as polyoxyethylene
sorbitan mono-oleate. The emulsions can also contain sweetening and
flavoring agents.
[0125] Pulmonary delivery of the cyclopropanated carbocyclic
2'-deoxynucleosides of preferred embodiments can also be employed.
The cyclopropanated carbocyclic 2'-deoxynucleoside is delivered to
the lungs while inhaling and traverses across the lung epithelial
lining to the blood stream. A wide range of mechanical devices
designed for pulmonary delivery of therapeutic products can be
employed, including but not limited to nebulizers, metered dose
inhalers, and powder inhalers, all of which are familiar to those
skilled in the art. These devices employ formulations suitable for
the dispensing of the cyclopropanated carbocyclic
2'-deoxynucleoside. Typically, each formulation is specific to the
type of device employed and can involve the use of an appropriate
propellant material, in addition to diluents, adjuvants, and/or
carriers useful in therapy.
[0126] The cyclopropanated carbocyclic 2'-deoxynucleoside and other
optional active ingredients are advantageously prepared for
pulmonary delivery in particulate form with an average particle
size of from 0.1 .mu.m or less to 10 .mu.m or more, more preferably
from about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, or 0.9 .mu.m to about
1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0,
7.5, 8.0, 8.5, 9.0, or 9.5 .mu.m. Pharmaceutically acceptable
carriers for pulmonary delivery of the cyclopropanated carbocyclic
2'-deoxynucleosides include carbohydrates such as trehalose,
mannitol, xylitol, sucrose, lactose, and sorbitol. Other
ingredients for use in formulations can include DPPC, DOPE, DSPC,
and DOPC. Natural or synthetic surfactants can be used, including
polyethylene glycol and dextrans, such as cyclodextran. Bile salts
and other related enhancers, as well as cellulose and cellulose
derivatives, and amino acids can also be used. Liposomes,
microcapsules, microspheres, inclusion complexes, and other types
of carriers can also be employed.
[0127] Pharmaceutical formulations suitable for use with a
nebulizer, either jet or ultrasonic, typically comprise the
cyclopropanated carbocyclic 2'-deoxynucleoside dissolved or
suspended in water at a concentration of about 0.01 or less to 100
mg or more of cyclopropanated carbocyclic 2'-deoxynucleoside per mL
of solution, preferably from about 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9,
or 10 mg to about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,
75, 80, 85, or 90 mg per mL of solution. The formulation can also
include a buffer and a simple sugar (e.g., for protein
stabilization and regulation of osmotic pressure). The nebulizer
formulation can also contain a surfactant, to reduce or prevent
surface induced aggregation of the cyclopropanated carbocyclic
2'-deoxynucleoside caused by atomization of the solution in forming
the aerosol.
[0128] Formulations for use with a metered-dose inhaler device
generally comprise a finely divided powder containing the active
ingredients suspended in a propellant with the aid of a surfactant.
The propellant can include conventional propellants, such as
chlorofluorocarbons, hydrochlorofluorocarbons, hydrofluorocarbons,
and hydrocarbons. Preferred propellants include
trichlorofluoromethane, dichlorodifluoromethane,
dichlorotetrafluoroethanol, 1,1,1,2-tetrafluoroethane, and
combinations thereof. Suitable surfactants include sorbitan
trioleate, soya lecithin, and oleic acid.
[0129] Formulations for dispensing from a powder inhaler device
typically comprise a finely divided dry powder containing the
cyclopropanated carbocyclic 2'-deoxynucleoside, optionally
including a bulking agent, such as lactose, sorbitol, sucrose,
mannitol, trehalose, or xylitol in an amount that facilitates
dispersal of the powder from the device, typically from about 1 wt.
% or less to 99 wt. % or more of the formulation, preferably from
about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 wt. % to about 55,
60, 65, 70, 75, 80, 85, or 90 wt. % of the formulation.
[0130] When the cyclopropanated carbocyclic 2'-deoxynucleoside is
administered by intravenous, cutaneous, subcutaneous, parenteral,
or other injection, it is preferably in the form of a pyrogen-free,
parenterally acceptable aqueous solution or oleaginous suspension.
Suspensions can be formulated according to methods well known in
the art using suitable dispersing or wetting agents and suspending
agents. The preparation of acceptable aqueous solutions with
suitable pH, isotonicity, stability, and the like, is within the
skill in the art. A preferred pharmaceutical composition for
injection preferably contains an isotonic vehicle such as
1,3-butanediol, water, isotonic sodium chloride solution, Ringer's
solution, dextrose solution, dextrose and sodium chloride solution,
lactated Ringer's solution, or other vehicles as are known in the
art. In addition, sterile fixed oils can be employed conventionally
as a solvent or suspending medium. For this purpose, any bland
fixed oil can be employed including synthetic mono or diglycerides.
In addition, fatty acids such as oleic acid can likewise be used in
the formation of injectable preparations. The pharmaceutical
compositions can also contain stabilizers, preservatives, buffers,
antioxidants, or other additives known to those of skill in the
art.
[0131] The duration of the injection can be adjusted depending upon
various factors, and can comprise a single injection administered
over the course of a few seconds or less, to 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24
hours or more of continuous intravenous administration.
[0132] The cyclopropanated carbocyclic 2'-deoxynucleosides can be
administered systematically or locally, via a liquid or gel, or as
an implant or device.
[0133] The anti-pox compositions of the preferred embodiments can
additionally employ adjunct components conventionally found in
pharmaceutical compositions in their art-established fashion and at
their art-established levels. Thus, for example, the compositions
can contain additional compatible pharmaceutically active materials
for combination therapy (such as supplementary antimicrobials,
antipruritics, astringents, local anesthetics, anti-inflammatory
agents, and the like), or can contain materials useful in
physically formulating various dosage forms of the preferred
embodiments, such as excipients, dyes, perfumes, thickening agents,
stabilizers, skin penetration enhancers, preservatives or
antioxidants.
[0134] Secondary skin infections are often associated with smallpox
infections. Accordingly, it is desirable to administer the
cyclopropanated carbocyclic 2'-deoxynucleosides of preferred
embodiments in preparations including other therapeutic agents such
as anti-microbial agents (anti-bacterials, anti-mycobacterials,
anti-virals, anti-fungal, anti-parasites, and the like). Examples
of anti-bacterials include beta-lactam antibiotics, penicillins
(such as natural penicillins, aminopenicillins,
penicillinase-resistant penicillins, carboxy penicillins, ureido
penicillins), cephalosporins (first generation, second generation,
and third generation cephalosporins), and other beta-lactams (such
as imipenem, monobactams), beta-lactamase inhibitors, vancomycin,
aminoglycosides and spectinomycin, tetracyclines, chloramphenicol,
erythromycin, lincomycin, clindamycin, rifampin, metronidazole,
polymyxins, sulfonamides and trimethoprim, and quinolines,
Acedapsone; Acetosulfone Sodium; Alamecin; Alexidine; Amdinocillin;
Amdinocillin Pivoxil; Amicycline; Amifloxacin; Amifloxacin
Mesylate; Amikacin; Amikacin Sulfate; Aminosalicylic acid;
Aminosalicylate sodium; Amoxicillin; Amphomycin; Ampicillin;
Ampicillin Sodium; Apalcillin Sodium; Apramycin; Aspartocin;
Astromicin Sulfate; Avilamycin; Avoparcin; Azithromycin;
Azlocillin; Azlocillin Sodium; Bacampicillin Hydrochloride;
Bacitracin; Bacitracin Methylene Disalicylate; Bacitracin Zinc;
Bambermycins; Benzoylpas Calcium; Berythromycin; Betamicin Sulfate;
Biapenem; Biniramycin; Biphenamine Hydrochloride; Bispyrithione
Magsulfex; Butikacin; Butirosin Sulfate; Capreomycin Sulfate;
Carbadox; Carbenicillin Disodium; Carbenicillin Indanyl Sodium;
Carbenicillin Phenyl Sodium; Carbenicillin Potassium; Carumonam
Sodium; Cefaclor; Cefadroxil; Cefamandole; Cefamandole Nafate;
Cefamandole Sodium; Cefaparole; Cefatrizine; Cefazaflur Sodium;
Cefazolin; Cefazolin Sodium; Cefbuperazone; Cefdinir; Cefepime;
Cefepime Hydrochloride; Cefetecol; Cefixime; Cefinenoxime
Hydrochloride; Cefinetazole; Cefinetazole Sodium; Cefonicid
Monosodium; Cefonicid Sodium; Cefoperazone Sodium; Ceforanide;
Cefotaxime Sodium; Cefotetan; Cefotetan Disodium; Cefotiam
Hydrochloride; Cefoxitin; Cefoxitin Sodium; Cefpimizole;
Cefpimizole Sodium; Cefpiramide; Cefpiramide Sodium; Cefpirome
Sulfate; Cefpodoxime Proxetil; Cefprozil; Cefroxadine; Cefsulodin
Sodium; Ceftazidime; Ceftibuten; Ceftizoxime Sodium; Ceftriaxone
Sodium; Cefuroxime; Cefuroxime Axetil; Cefuroxime Pivoxetil;
Cefuroxime Sodium; Cephacetrile Sodium; Cephalexin; Cephalexin
Hydrochloride; Cephaloglycin; Cephaloridine; Cephalothin Sodium;
Cephapirin Sodium; Cephradine; Cetocycline Hydrochloride;
Cetophenicol; Chloramphenicol; Chloramphenicol Palmitate;
Chloramphenicol Pantothenate Complex; Chloramphenicol Sodium
Succinate; Chlorhexidine Phosphanilate; Chloroxylenol;
Chlortetracycline Bisulfate; Chlortetracycline Hydrochloride;
Cinoxacin; Ciprofloxacin; Ciprofloxacin Hydrochloride; Cirolemycin;
Clarithromycin; Clinafloxacin Hydrochloride; Clindamycin;
Clindamycin Hydrochloride; Clindamycin Palmitate Hydrochloride;
Clindamycin Phosphate; Clofazimine; Cloxacillin Benzathine;
Cloxacillin Sodium; Cloxyquin; Colistimethate Sodium; Colistin
Sulfate; Coumermycin; Coumermycin Sodium; Cyclacillin; Cycloserine;
Dalfopristin; Dapsone; Daptomycin; Demeclocycline; Demeclocycline
Hydrochloride; Demecycline; Denofungin; Diaveridine; Dicloxacillin;
Dicloxacillin Sodium; Dihydrostreptomycin Sulfate; Dipyrithione;
Dirithromycin; Doxycycline; Doxycycline Calcium; Doxycycline
Fosfatex; Doxycycline Hyclate; Droxacin Sodium; Enoxacin;
Epicillin; Epitetracycline Hydrochloride; Erythromycin;
Erythromycin Acistrate; Erythromycin Estolate; Erythromycin
Ethylsuccinate; Erythromycin Gluceptate; Erythromycin Lactobionate;
Erythromycin Propionate; Erythromycin Stearate; Ethambutol
Hydrochloride; Ethionamide; Fleroxacin; Floxacillin; Fludalanine;
Flumequine; Fosfomycin; Fosfomycin Tromethamine; Fumoxicillin;
Furazolium Chloride; Furazolium Tartrate; Fusidate Sodium; Fusidic
Acid; Gentamicin Sulfate; Gloximonam; Gramicidin; Haloprogin;
Hetacillin; Hetacillin Potassium; Hexedine; Ibafloxacin; Imipenem;
Isoconazole; Isepamicin; Isoniazid; Josamycin; Kanamycin Sulfate;
Kitasamycin; Levofuraltadone; Levopropylcillin Potassium;
Lexithromycin; Lincomycin; Lincomycin Hydrochloride; Lomefloxacin;
Lomefloxacin Hydrochloride; Lomefloxacin Mesylate; Loracarbef;
Mafenide; Meclocycline; Meclocycline Sulfosalicylate; Megalomicin
Potassium Phosphate; Mequidox; Meropenem; Methacycline;
Methacycline Hydrochloride; Methenamine; Methenamine Hippurate;
Methenamine Mandelate; Methicillin Sodium; Metioprim; Metronidazole
Hydrochloride; Metronidazole Phosphate; Meziocillin; Meziocillin
Sodium; Minocycline; Minocycline Hydrochloride; Mirincamycin
Hydrochloride; Monensin; Monensin Sodium; Nafcillin Sodium;
Nalidixate Sodium; Nalidixic Acid; Natamycin; Nebramycin; Neomycin
Palmitate; Neomycin Sulfate; Neomycin Undecylenate; Netilmicin
Sulfate; Neutramycin; Nifuradene; Nifuraldezone; Nifuratel;
Nifuratrone; Nifurdazil; Nifurimide; Nifurpirinol; Nifurquinazol;
Nifurthiazole; Nitrocycline; Nitrofurantoin; Nitromide;
Norfloxacin; Novobiocin Sodium; Ofloxacin; Ornetoprim; Oxacillin
Sodium; Oximonam; Oximonam Sodium; Oxolinic Acid; Oxytetracycline;
Oxytetracycline Calcium; Oxytetracycline Hydrochloride; Paldimycin;
Parachlorophenol; Paulomycin; Pefloxacin; Pefloxacin Mesylate;
Penamecillin; Penicillin G Benzathine; Penicillin G Potassium;
Penicillin G Procaine; Penicillin G Sodium; Penicillin V;
Penicillin V Benzathine; Penicillin V Hydrabamine; Penicillin V
Potassium; Pentizidone Sodium; Phenyl Aminosalicylate; Piperacillin
Sodium; Pirbenicillin Sodium; Piridicillin Sodium; Pirlimycin
Hydrochloride; Pivampicillin Hydrochloride; Pivampicillin Pamoate;
Pivampicillin Probenate; Polymyxin B Sulfate; Porfiromycin;
Propikacin; Pyrazinamide; Pyrithione Zinc; Quindecamine Acetate;
Quinupristin; Racephenicol; Ramoplanin; Ranimycin; Relomycin;
Repromicin; Rifabutin; Rifametane; Rifamexil; Rifamide; Rifampin;
Rifapentine; Rifaximin; Rolitetracycline; Rolitetracycline Nitrate;
Rosaramicin; Rosaramicin Butyrate; Rosaramicin Propionate;
Rosaramicin Sodium Phosphate; Rosaramicin Stearate; Rosoxacin;
Roxarsone; Roxithromycin; Sancycline; Sanfetrinem Sodium;
Sarmoxicillin; Sarpicillin; Scopafungin; Sisomicin; Sisomicin
Sulfate; Sparfloxacin; Spectinomycin Hydrochloride; Spiramycin;
Stallimycin Hydrochloride; Steffimycin; Streptomycin Sulfate;
Streptonicozid; Sulfabenz; Sulfabenzamide; Sulfacetamide;
Sulfacetamide Sodium; Sulfacytine; Sulfadiazine; Sulfadiazine
Sodium; Sulfadoxine; Sulfalene; Sulfamerazine; Sulfameter;
Sulfamethazine; Sulfamethizole; Sulfamethoxazole;
Sulfamonomethoxine; Sulfamoxole; Sulfanilate Zinc; Sulfanitran;
Sulfasalazine; Sulfasomizole; Sulfathiazole; Sulfazamet;
Sulfisoxazole; Sulfisoxazole Acetyl; Sulfisoxazole Diolamine;
Sulfomyxin; Sulopenem; Sultamicillin; Suncillin Sodium;
Talampicillin Hydrochloride; Teicoplanin; Temafloxacin
Hydrochloride; Temocillin; Tetracycline; Tetracycline
Hydrochloride; Tetracycline Phosphate Complex; Tetroxoprim;
Thiamphenicol; Thiphencillin Potassium; Ticarcillin Cresyl Sodium;
Ticarcillin Disodium; Ticarcillin Monosodium; Ticlatone; Tiodonium
Chloride; Tobramycin; Tobramycin Sulfate; Tosufloxacin;
Trimethoprim; Trimethoprim Sulfate; Trisulfapyrimidines;
Troleandomycin; Trospectomycin Sulfate; Tyrothricin; Vancomycin;
Vancomycin Hydrochloride; Virginiamycin; Zorbamycin.
Anti-mycobacterials include Myambutol (Ethambutol Hydrochloride),
Dapsone (4,4'-diaminodiphenylsulfone), Paser Granules
(aminosalicylic acid granules), Priftin (rifapentine),
Pyrazinamide, Isoniazid, Rifadin (Rifampin), Rifadin IV, Rifamate
(Rifampin and Isoniazid), Rifater (Rifampin, Isoniazid, and
Pyrazinamide), Streptomycin Sulfate and Trecator-SC (Ethionamide).
Anti-virals include amantidine and rimantadine, ribivarin,
acyclovir, vidarabine, trifluorothymidine, ganciclovir, zidovudine,
retinovir, interferons, Acemannan; Acyclovir; Acyclovir Sodium;
Adefovir; Alovudine; Alvircept Sudotox; Amantadine Hydrochloride;
Aranotin; Arildone; Atevirdine Mesylate; Avridine; Cidofovir;
Cipamfylline; Cytarabine Hydrochloride; Delavirdine Mesylate;
Desciclovir; Didanosine; Disoxaril; Edoxudine; Enviradene;
Enviroxime; Famciclovir; Famotine Hydrochloride; Fiacitabine;
Fialuridine; Fosarilate; Foscarnet Sodium; Fosfonet Sodium;
Ganciclovir; Ganciclovir Sodium; Idoxuridine; Kethoxal; Lamivudine;
Lobucavir; Memotine Hydrochloride; Methisazone; Nevirapine;
Penciclovir; Pirodavir; Ribavirin; Rimantadine Hydrochloride;
Saquinavir Mesylate; Somantadine Hydrochloride; Sorivudine;
Statolon; Stavudine; Tilorone Hydrochloride; Trifluridine;
Valacyclovir Hydrochloride; Vidarabine; Vidarabine Phosphate;
Vidarabine Sodium Phosphate; Viroxime; Zalcitabine; Zidovudine;
Zinviroxime and integrase inhibitors. Anti-fungals include
imidazoles and triazoles, polyene macrolide antibiotics,
griseofulvin, amphotericin B, and flucytosine. Antiparasites
include heavy metals, antimalarial quinolines, folate antagonists,
nitroimidazoles, benzimidazoles, avermectins, praxiquantel,
ornithine decarboxylase inhibitors, phenols (e.g., bithionol,
niclosamide); synthetic alkaloid (e.g., dehydroemetine);
piperazines (e.g., diethylcarbamazine); acetanilide (e.g.,
diloxanide furonate); halogenated quinolines (e.g., iodoquinol
(diiodohydroxyquin)); nitrofurans (e.g., nifurtimox); diamidines
(e.g., pentamidine); tetrahydropyrimidine (e.g., pyrantel pamoate);
sulfated naphthylamine (e.g., suramin).
[0135] Preferred anti-infectives for use in combination with the
cyclopropanated carbocyclic 2'-deoxynucleosides of preferred
embodiments include Difloxacin Hydrochloride; Lauryl Isoquinolinium
Bromide; Moxalactam Disodium; Omidazole; Pentisomicin; Sarafloxacin
Hydrochloride; Protease inhibitors of HIV and other retroviruses;
Integrase Inhibitors of HIV and other retroviruses; Cefaclor
(Ceclor); Acyclovir (Zovirax); Norfloxacin (Noroxin); Cefoxitin
(Mefoxin); Cefuroxime axetil (Ceftin); Ciprofloxacin (Cipro);
Aminacrine Hydrochloride; Benzethonium Chloride: Bithionolate
Sodium; Bromchlorenone; Carbamide Peroxide; Cetalkonium Chloride;
Cetylpyridinium Chloride: Chlorhexidine Hydrochloride; Clioquinol;
Domiphen Bromide; Fenticlor; Fludazonium Chloride; Fuchsin, Basic;
Furazolidone; Gentian Violet; Halquinols; Hexachlorophene: Hydrogen
Peroxide; Ichthammol; Imidecyl Iodine; Iodine; Isopropyl Alcohol;
Mafenide Acetate; Meralein Sodium; Mercufenol Chloride; Mercury,
Ammoniated; Methylbenzethonium Chloride; Nitrofurazone;
Nitromersol; Octenidine Hydrochloride; Oxychlorosene; Oxychlorosene
Sodium; Parachlorophenol, Camphorated; Potassium Permanganate;
Povidone-Iodine; Sepazonium Chloride; Silver Nitrate; Sulfadiazine,
Silver; Symclosene; Thimerfonate Sodium; Thimerosal; and Troclosene
Potassium.
[0136] When the cyclopropanated carbocyclic 2'-deoxynucleoside is
employed for the prevention or treatment of poxvirus infection, it
is particularly preferred to administer it in combination with
other agents employed to treat poxvirus infection, such as
cidofovir, and the like. The cyclopropanated carbocyclic
2'-deoxynucleoside can also be administered in conjunction with a
smallpox vaccine (e.g., the vaccinia vaccine--a live preparation of
the infectious vaccinia virus that does not contain the smallpox
virus) in those instances wherein the vaccine is as a precaution
against developing the disease after exposure to infection, or in
conjunction with Vaccinia Immune Globulin, used to treat
complications of the smallpox vaccine.
[0137] The cyclopropanated carbocyclic 2'-deoxynucleoside can be
provided to an administering physician or other health care
professional in the form of a kit. The kit is a package which
houses a container which contains the cyclopropanated carbocyclic
2'-deoxynucleoside in suitable form and instructions for
administering the pharmaceutical composition to a subject. The kit
can optionally also contain one or more additional therapeutic
agents. The kit can optionally contain one or more diagnostic tools
and instructions for use. For example, a kit containing a
composition comprising a cyclopropanated carbocyclic
2'-deoxynucleoside in combination with one or more additional
therapeutic agents, such as antiviral, antibacterial, and/or
anti-infective agents, can be provided, or separate pharmaceutical
compositions containing a cyclopropanated carbocyclic
2'-deoxynucleoside and additional therapeutic agents can be
provided. The kit can also contain separate doses of the
cyclopropanated carbocyclic 2'-deoxynucleoside for serial or
sequential administration. The kit can contain suitable delivery
devices, e.g., syringes, inhalation devices, and the like, along
with instructions for administrating the cyclopropanated
carbocyclic 2'-deoxynucleoside and any other therapeutic agent. The
kit can optionally contain instructions for storage, reconstitution
(if applicable), and administration of any or all therapeutic
agents included. The kits can include a plurality of containers
reflecting the number of administrations to be given to a subject.
In a particularly preferred embodiment, a kit for the treatment of
smallpox infection is provided that includes a cyclopropanated
carbocyclic 2'-deoxynucleoside and one or more of smallpox vaccine,
Vaccinia Immune Globulin, cidofovir, or one or more antibiotic
formulations for treating secondary skin infections. The kit can
also include instructions, an assay, or a diagnostic for
determining if a patient has been exposed to smallpox virus or has
an active smallpox infection, or to diagnose or characterize a
secondary skin infection or other condition associated with
smallpox infection.
[0138] The cyclopropanated carbocyclic 2'-deoxynucleosides of
preferred embodiments can be administered prophylactically for the
prevention of smallpox infection in exposed individuals.
Alternatively, therapy is preferably initiated as early as possible
following the onset of signs and symptoms of smallpox infection.
The administration route, amount administered, and frequency of
administration will vary depending on the age of the patient,
condition to be treated, and severity of the condition.
Contemplated amounts, dosages, and routes of administration for
smallpox infections are similar to those established for the
treatment of other viral infections using commercially available
antiviral agents. Detailed information relating to administration
and dosages of antiviral agents can be found in the Physician's
Desk Reference, 47th edition, pp. 844-850, 1993 and in Hayden et
al., "Antiviral Agents" in Basic Principles in the Diagnosis of
Infectious Diseases, pp. 271-274. This information can be adapted
in designing treatment regimes utilizing the cyclopropanated
carbocyclic 2'-deoxynucleosides of preferred embodiments.
[0139] Contemplated amounts of cyclopropanated carbocyclic
2'-deoxynucleosides for oral administration to treat smallpox
infections are from about 10 mg or less to about 2000 mg or more
administered from about every 4 hours or less to about every 6
hours or more (or from about 4 times daily to about 6 times daily)
for from about 5 days or less to about 10 days or more (40 mg/day
or less to about 15,000 mg/day or more) or until there is a
significant improvement in the condition. To prevent or inhibit the
onset of infection in susceptible or exposed individuals, doses of
from about 10 mg or less to about 1000 mg or more are orally
administered once, twice, or multiple times a day, typically for up
to about 12 months, or, in certain circumstances, indefinitely
(from about 10 mg/day to about 1,000 mg/day). When treatment is
long term, it can be desirable to vary the dosage, employing a
higher dosage early in the treatment, and a lower dosage later in
the treatment. For topical administration to skin rash or lesions
associated with smallpox, a topical preparation containing from
about 10 mg or less to about 100 mg or more cyclopropanated
carbocyclic 2'-deoxynucleoside per gram of preparation is typically
applied in an amount sufficient to adequately cover all lesions, or
lesions on only selected areas of the body. Higher or lower dosages
can be desirable, depending upon the patient being treated. The
topical preparation is applied every three to six hours from four
to six times a day for about 5 days or less to 10 days or more or
until the lesions have scabbed over (from about 100 mg/day or less
to about 1,000 mg/day or more). The dose size per application can
be adjusted depending upon the total affected area, but preferably
approximates a one cubic centimeter ribbon of preparation per
sixteen square centimeters of skin surface area. For intravenous
administration, from about 1 mg/kg to about 10 mg/kg is infused at
a constant rate over 30 minutes or less to about 1 hour, 2 hours or
more, every 6 hours or less to 8 hours or more (typically, from
about 3 mg/kg/day to about 30 mg/kg/day) for about 5 days or less
to about 7 days or more.
Experiment #1
General Approach for Determining Antiviral Activity and
Toxicity
[0140] The cyclopropanated carbocyclic 2'-deoxynucleosides of
preferred embodiments were tested for toxicity and antiviral
activity against orthopoxviruses using the methodology developed by
E. Kern, Ph.D., University of Alabaina Birmingham (see, e.g.,
Prichard, et al., Antimicrobial Agents and Chemotherapy, April
2006, p. 1336-1341; Prichard, et al., Antiviral Research, received
Jul. 25, 2005, accepted Jan. 19, 2006, article in press). The
experimental approach was based upon the following: 1) an
inexpensive, rapid assay such as a cytopathic effect inhibition
assay that is semi-automated was used initially to screen out the
negatives; 2) all screening assays were conducted in low-passaged
human cells; 3) each assay system contained cidofovir (CDV) as a
positive control and acyclovir (ACV) as a negative control; 4)
efficacy was preferably demonstrated by at least two different
assay systems that detect functional biologic activity; 5) efficacy
against vaccinia virus and cowpox virus was preferably confirmed
using other isolates; 6) toxicity was determined using both resting
and proliferating human fibroblast cells; and 7) for selected
compounds, toxicity in rodent myeloid and erythroid progenitor
cells was assessed.
Screening Assay Systems for Determining Antiviral Activity against
Vaccinia Virus and Cowpox Virus
[0141] Experimental compounds were initially screened for activity
using the CPE assay in HFF cells. Further testing in two other
cells lines, Vero and MRC-5, and against other strains of virus was
conducted for selected compounds that demonstrated activity in
other assay systems. All of the screening assay systems utilized
were selected to show specific inhibition of a biologic function,
i.e., cytopathic effect in susceptible human cells. In the
CPE-inhibition assay, the experimental compound was added 1 hr
prior to infection so that the assay system would have maximum
sensitivity and detect inhibitors of early replicative steps, such
as adsorption or penetration, as well as later events. To rule out
non-specific inhibition of virus binding to cells, all experimental
compounds that showed reasonable activity in the CPE assay were
confirmed using a classical plaque reduction assay in which the
experimental compound was added 1 hr after infection. These assay
systems also can be manipulated by increasing the pre-treatment
time in order to demonstrate antiviral activity with
oligodeoxynucleotides and/or peptides. By delaying the time of
addition of experimental compound after infection, information
regarding which step in the virus life cycle is inhibited (i.e.,
early vs. late functions) can be gained. A direct inactivation
assay can also be employed to determine the virucidal activity of
selected experimental compounds.
[0142] Efficacy
[0143] In all the assays used for primary screening, a minimum of
six experimental compound concentrations was used covering a range
of 100 .mu.g/ml to 0.03 .mu.g/ml, in 5-fold increments. These data
permitted acceptable dose response curves to be obtained. From
these data, the dose that inhibited viral replication by 50%
(effective concentration 50; EC.sub.50) was calculated using the
computer software program MacSynergy II by M. N. Prichard, K. R.
Asaltine, and C. Shipman, Jr., University of Michigan, Ann Arbor,
Mich.
[0144] Toxicity
[0145] The same experimental compound concentrations used to
determine efficacy were also used on uninfected cells in each assay
to determine toxicity of each experimental compound. The
experimental compound concentration that was cytotoxic to cells as
determined by their failure to take up a vital stain, neutral red,
(cytotoxic concentration 50; CC.sub.50) was determined as described
above. The neutral red uptake assay was reliable and reproducible
and allowed quantitation of toxicity based on the number of viable
cells rather than cellular metabolic activity. A cell proliferation
assay using HFF cells is a very sensitive assay for detecting
experimental compound toxicity to dividing cells and for
determining the experimental compound concentration that inhibits
cell growth by 50% (IC.sub.50) is calculated as described above. In
comparison with four human diploid cell lines and Vero cells, HFF
cells are the most sensitive and predictive of toxicity for bone
marrow cells.
[0146] Assessment of Drug Activity
[0147] To determine if each experimental compound has a sufficient
antiviral activity that exceeds its level of toxicity, a
selectivity index (SI) was calculated according to
CC.sub.50/EC.sub.50. This index, also referred to as a therapeutic
index, was used to determine if a compound warranted further study.
For these studies, a compound that had an SI of 10 or greater was
evaluated in additional assay systems.
Confirmation of Antiviral Activity and Toxicity for Vaccinia Virus
and Cowpox Virus
[0148] Assay
[0149] Experimental compounds that showed activity in the
CPE-inhibition assay were confirmed using the plaque reduction
assay. Descriptions of the plaque reduction assay are provided in
Keith et al., Antimicrobial Agents and Chemotherapy, July 2003, p.
2193-2198; and Keith et al., Antimicrobial Agents and Chemotherapy,
May 2004, p. 1869-1871. Susceptibility of additional virus strains
of vaccinia virus and activity in other cell types was also
determined for selected experimental compounds.
[0150] Toxicity
[0151] In addition to the toxicity component incorporated into each
assay system, a standardized cell cytotoxicity assay using a vital
stain uptake (Neutral Red) was performed using 7 days of drug
exposure to confluent non-dividing cells. A cell proliferation
assay using HFF cells is a very sensitive assay for detecting drug
toxicity to dividing cells and the drug concentration that inhibits
cell growth by 50% (IC.sub.50) was calculated as described
above.
Laboratory Procedures for Determining Antiviral Efficacy and
Toxicity
[0152] Preparation of Experimental Compounds for In Vitro
Testing
[0153] The experimental compound was weighed using an analytical
balance and reconstituted in the appropriate vehicle. If the
compound is water soluble, it is dissolved in tissue culture medium
without serum at 1 mg/ml and diluted for use as indicated below in
the description of the assay system. If the compound is not water
soluble, then it is automatically dissolved in DMSO at a
concentration of 10 mg/ml and diluted for use in each assay. When
DMSO or other solvents are used, control cultures receive media
containing the same concentration of solvent as test cultures.
Compounds that are not sufficiently soluble are generally not
preferred for use as antiviral agents; however, they can be
modified chemically to increase their solubility.
[0154] Screening and Confirmation Assays for Vaccinia Virus and
Cowpox Virus
[0155] Preparation of Human Foreskin Fibroblast (HFF) Cells
[0156] Newborn human foreskins were obtained as soon as possible
after circumcision and placed in minimal essential medium (MEM)
containing vancomycin, fungizone, penicillin, and gentamicin at the
usual concentrations, for 4 h. The medium was then removed, the
foreskin minced into small pieces and washed repeatedly with
phosphate buffered saline (PBS) deficient in calcium and magnesium
(PD) until red cells were no longer present. The tissue was then
trypsinized using trypsin at 0.25% with continuous stirring for 15
min at 37.degree. C. in a CO.sub.2 incubator. At the end of each 15
min period, the tissue was allowed to settle to the bottom of the
flask. The supernatant containing cells was poured through sterile
cheesecloth into a flask containing MEM and 10% fetal bovine serum.
The flask containing the medium was kept on ice throughout the
trypsinizing procedure. After each addition of cells, the
cheesecloth was washed with a small amount of MEM containing serum.
Fresh trypsin was added each time to the foreskin pieces and the
procedure repeated until all the tissue was digested. The
cell-containing medium was then centrifuged at 1000 RPM at
4.degree. C. for 10 min. The supernatant liquid was discarded and
the cells resuspended in a small amount of MEM with 10% fetal
bovine serum (FBS). The cells were then placed in an appropriate
number of 25 cm.sup.2 tissue culture flasks. As cells became
confluent and needed trypsinization, they were expanded into larger
flasks. The cells were kept on vancomycin and fungizone to passage
four, and maintained on penicillin and gentamicin. Cells were used
only through passage 10.
[0157] Cytopathic Effect Inhibition Assay
[0158] Low passage HFF cells were seeded into 96 well tissue
culture plates 24 h prior to use at a cell concentration of
2.5.times.10.sup.5 cells per ml in 0.1 ml of MEM supplemented with
10% FBS. The cells were then incubated for 24 h at 37.degree. C. in
a CO.sub.2 incubator. After incubation, the medium was removed and
125 .mu.l of experimental drug was added to the first row in
triplicate wells, all other wells having 100 .mu.l of MEM
containing 2% FBS. The experimental compound in the first row of
wells was then diluted serially 1:5 throughout the remaining wells
by transferring 25 .mu.l using the BioMek 2000 Laboratory
Automation Workstation. After dilution of the experimental
compound, 100 .mu.l of the appropriate virus concentration was
added to each well, excluding cell control wells, which received
100 .mu.l of MEM. The virus concentration utilized was 1000 PFUs
per well. The plates were then incubated at 37.degree. C. in a
CO.sub.2 incubator for 7 days. After the incubation period, media
was aspirated and the cells stained with a 0.1% crystal violet in
3% formalin solution for 4 h. The stain was removed and the plates
were rinsed using tap water until all excess stain was removed. The
plates were allowed to dry for 24 h and then read on a BioTek
Multiplate Autoreader at 620 nm. The EC.sub.50 values were
determined by comparing experimental compound treated and untreated
cells using a computer program.
[0159] Plaque Reduction Assay Using Semi-Solid Overlay
[0160] Two days prior to use, HFF cells were plated into 6 well
plates and incubated at 37.degree. C. with 5% CO.sub.2 and 90%
humidity. On the date of assay, the experimental compound was made
up at twice the desired concentration in 2.times.MEM and then
serially diluted 1:5 in 2.times.MEM using 6 concentrations of
experimental compound. The initial starting concentration was
usually 200 .mu.g/ml down to 0.06 .mu.g/ml. The virus to be used
was diluted in MEM containing 10% FBS to a desired concentration
which gave 20-30 plaques per well. The media was then aspirated
from the wells and 0.2 ml of virus was added to each well in
duplicate with 0.2 ml of media being added to drug toxicity wells.
The plates were then incubated for 1 h with shaking every 15 min.
After the incubation period, an equal amount of 1% agarose was
added to an equal volume of each drug dilution. This gives final
experimental compound concentrations beginning with 100 .mu.g/ml
and ending with 0.03 .mu.g/ml and a final agarose overlay
concentration of 0.5%. The drug/agarose mixture was applied to each
well in 2 ml volume and the plates were incubated for 3 days, after
which the cells were stained with a 0.01% solution of neutral red
in phosphate buffered saline. After a 5-6 h incubation period, the
stain was aspirated, and plaques counted using a stereomicroscope
at 10.times. magnification.
[0161] Screening and Confirmation Assays for Toxicity
[0162] Neutral Red Uptake Assay
[0163] Twenty-four hours prior to assay, HFF cells were plated into
96 well plates at a concentration of 2.5.times.10.sup.4 cells per
well. After 24 h, the media was aspirated and 125 .mu.l of
experimental compound was added to the first row of wells and then
diluted serially 1:5 using the BioMek 2000 Laboratory Automation
Workstation in a manner similar to that used in the CPE assay.
After drug addition, the plates were incubated for 7 days in a
CO.sub.2 incubator at 37.degree. C. At this time, the
media/experimental compound was aspirated and 200 .mu.l/well of
0.01% neutral red in PBS was added. This was incubated in the
CO.sub.2 incubator for 1 h. The dye was aspirated and the cells
were washed using a Nunc Plate Washer. After removing the PBS, 200
.mu.g/well of 50% ETOH/1% glacial acetic acid (in H.sub.2O) was
added. The plates were rotated for 15 min and the optical densities
read at 540 nm on a plate reader. The EC.sub.50 values were
determined by comparing experimental compound treated and untreated
cells using a computer program.
[0164] Cell Proliferation Assay
[0165] Twenty-four hours prior to assay, HFF cells were seeded in
6-well plates at a concentration of 2.5.times.10.sup.4 cells per
well in MEM containing 10% FBS. On the day of the assay, the
experimental compound was diluted serially in MEM containing 10%
FBS at increments of 1:5 covering a range from 100 .mu.g/ml to 0.03
.mu.g/ml. For experimental compounds that have to be solubilized in
DMSO, control wells receive MEM containing 1% DMSO. The media from
the wells was aspirated and 2 ml of each experimental compound was
then added to each well. The cells were incubated in a CO.sub.2
incubator at 37.degree. C. for 72 h. At the end of this time, the
media-drug solution was removed and the cells washed. One ml of
0.25% trypsin was added to each well and incubated until the cells
started to come off of the plate. The cell-media mixture was then
pipetted up and down vigorously to break up the cell suspension and
0.2 ml of the mixture was added to 9.8 ml of Isoton III and counted
using a Coulter Counter. Each sample was counted 3 times with 2
replicate wells per sample.
[0166] Bone Marrow Clonogenic Assays
[0167] In vitro toxicity to bone marrow progenitor cells can be
determined by inhibition of myeloid (colony-forming units
granulocyte/macrophage (CFU-GM)) and erythroid (burst-forming
unit-erythroid (BFU-E)) colony formation in soft agar clonal
assays. Using a 21-23 gauge needle attached to a syringe, rodent
bone marrow cells are collected from the leg bone of rats or mice
by flushing with Isocoves' Modified Dulbecco's medium (IMDM). A
single cell suspension is obtained by repeated aspiration through
the needle. Nucleated cells are enumerated with a hemacytometer and
adjusted to the desired cell concentration in IMDM. Murine CFU-GM
assays are prepared with 2.5.times.10.sup.5 nucleated cells/ml, 20%
FBS, 10 ng/ml rmGM-CSF, and 0.2% agarose. BFU-E cultures include
30% FBS, 1% deionized BSA, 0.1 mM 2-ME, 4 U/ml rhEpo, 10 ng/ml
rmIL-3, 2.5.times.105 nucleated cells/ml and 0.2% agarose.
Triplicate wells (in 6 well plates) containing 0.1 ml of
experimental compound (10.times.) receive 1 ml of either culture
mixture for each concentration group and slowly mixed. The cultures
are allowed to gel at 4.degree. C. and then incubated for 7
(CFU-GM) or 9 (BFU-E) days at 37.degree. C. in a humidified
atmosphere of 5% CO.sub.2 in air. Colonies are counted using an
inverted microscope. CFU-GM colonies are identified as cell clones
containing at least 40 cells. BFU-E cultures are stained with
dianisidine, and aggregates of greater than 60
hemoglobin-containing cells are counted as erythroid colonies. The
median inhibitory concentration (IC.sub.50) and the 90% inhibitory
concentration (IC.sub.90) are derived from linear regression
analysis of the logarithm of drug concentration versus CFU-GM or
BFU-E survival fraction.
Plaque Reduction and Cytopathic Effect Inhibition Assay Results
[0168] Assays were conducted as described above to directly measure
the extent to which N-MCT, N-MCBrU, and CVD (a positive control
drug) inhibited the effects of infection by vaccinia virus
(Copenhagen Strain) and cowpox virus (Brighton strain) in Human
Foreskin Fibroblast (HFF) tissue culture. Results of the testing
(Table 1) indicated that N-MCT exhibited superior antiviral potency
compared to CDV. N-MCT was more active than M-MCBrU, and that
M-MCBrU was more active than CVD. Activity in the plaque assay for
N-MCT was approximately 7-fold higher than for CVD. All compounds
tested were non-cytotoxic.
TABLE-US-00001 TABLE 1 Vaccinia Cowpox (Copenhagen) (Brighton)
CC.sub.50 EC.sub.50 EC.sub.50 Drug Assay (.mu.g/ml) (.mu.g/ml) SI
(.mu.g/ml) SI N-MCT CPE >100 0.73 >137 0.31 >323 N-MCBrU
CPE >100 0.6 >156 11.5 >8.5 CDV CPE >100 1.8 >56 2.1
>48 N-MCT Plaque reduction >100 0.46 >217 0.6 >167
N-MCT Plaque reduction >100 2.6 >38.5 3.4 >29.4 CDV Plaque
reduction >100 3.5 >29 4.1 >25 EC.sub.50 (.mu.g/ml) (50%
Effective Concentration) = concentration required to inhibit
virus-induced effect by 50% CC.sub.50 (50% Cytotoxic Concentration)
= concentration required to cause cytotoxic effect by 50% SI
(Selective Index) = CC.sub.50/EC.sub.50
BALB/c Mice Inoculated with Vaccinia IHD
[0169] The effect of twice daily i.p. treatment with N-MCT
(compared to placebo or CDV) on the mortality of BALB/c mice
inoculated intranasally with vaccinia IHD strain was investigated.
N-MCT was prepared in 0.4% CMC and delivered i.p. in 0.1 ml doses.
CDV was prepared in sterile saline and given i.p. in 0.1 ml doses.
Animals were treated twice daily for 5 days beginning 24 hours post
viral inoculation. Results of the testing (Table 2) indicated that
N-MCT exhibited similar in vivo antiviral potency compared to CDV.
Because of the reduced toxicity of N-MCT compared to CVD, higher
dosages were administered without increased mortality.
TABLE-US-00002 TABLE 2 Mortality Mean Day Treatment Number Percent
P-value of Death P-value Placebo - Saline 15/15 100 -- 7.6 -- CDV
(15 mg/kg) 0/15 0 <0.001 -- -- N-MCT (50 mg/kg) 0/15 0 <0.001
-- -- N-MCT (16.7 mg/kg) 0/15 0 <0.001 -- -- N-MCT (5.6 mg/kg)
3/15 20 <0.001 8.7 <0.05
BALB/c Mice Inoculated with Cowpox Brighton Strain
[0170] The effect of twice daily i.p. treatment with N-MCT
(compared to placebo or CDV) on the mortality of BALB/c mice
inoculated intranasally with cowpox was investigated. N-MCT was
prepared in 0.4% CMC and delivered i.p. in 0.1 ml doses. CDV was
prepared in sterile saline and given i.p. in 0.1 ml doses. Animals
were treated twice daily for 5 days beginning 24 hours post viral
inoculation. Results of the testing (Table 3) indicated that N-MCT
exhibited satisfactory in vivo antiviral potency slightly less than
that of CDV. Because of the reduced toxicity of N-MCT compared to
CVD, higher dosages were administered without increased
mortality.
TABLE-US-00003 TABLE 3 Mortality Mean Day Treatment Number Percent
P-value of Death P-value Placebo - Saline 15/15 100 -- 9.6 -- CDV
(15 mg/kg) 0/15 0 <0.01 -- -- N-MCT (50 mg/kg) 2/15 13 <0.001
7.5 Not Significant N-MCT 3/15 20 <0.001 13.3 0.01 (16.7 mg/kg)
N-MCT (5.6 mg/kg) 6/15 40 <0.001 14.0 0.05
BALB/c Mice Inoculated with Cowpox Brighton Strain
[0171] The effect of twice daily i.p. treatment with N-MCT
(compared to placebo or CDV) on the mortality of BALB/c mice
challenged with cowpox was investigated. N-MCT was prepared in 0.4%
CMC and delivered i.p. in 0.1 ml doses to treat the resulting
infection. CDV was prepared in sterile saline and given i.p. in 0.1
ml doses. Animals were treated twice daily for 7 days beginning 1
day after viral challenge. Results of the testing (Table 4)
indicated that N-MCT exhibited similar in vivo antiviral potency
compared to CDV.
TABLE-US-00004 TABLE 4 Treatment Number of Survivors Placebo -
Saline 0/10 CDV (100 mg/kg/day) 10/10 N-MCT (100 mg/kg/day) 9/10
N-MCT (30 mg/kg/day) 2/10 N-MCT (10 mg/kg/day) 1/10
BALB/c Mice Inoculated with Vaccinia WR Strain
[0172] The effect of twice daily i.p. treatment with N-MCT
(compared to placebo or CDV) on the mortality of BALB/c mice
challenged with vaccinia WR strain, a more virulent strain than the
vaccinia IHD strain, was investigated. N-MCT was prepared in 0.4%
CMC and delivered i.p. in 0.1 ml doses to treat the resulting
infection. CDV was prepared in sterile saline and given i.p. in 0.1
ml doses. Animals were treated twice daily for 7 days beginning 1
day after viral challenge. Results of the testing (Table 5)
indicated that N-MCT did not exhibit efficacy against the vaccinia
WR strain.
TABLE-US-00005 TABLE 5 Treatment Number of Survivors Placebo -
Saline 0/20 CDV (100 mg/kg/day) 10/10 N-MCT (100 mg/kg/day) 0/10
N-MCT (30 mg/kg/day) 0/10 N-MCT (10 mg/kg/day) 0/10
[0173] The above described experiments were repeated. The results
of the experiments regarding activity of N-MCT against a severe
vaccinia (WR strain) respiratory infection in mice (high virus
challenge of 105 PFU/mouse) is presented in Table 6.
TABLE-US-00006 TABLE 6 Compound Treatment Survivors/ Mean Lung
(mg/kg/day).sup.a Days Total MDD.sup.b Virus Titer.sup.c N-MCT 1-7
7/10*** 8.3 .+-. 0.6 7.9 .+-. 0.2** (100 mg/kg/day) N-MCT 1-7 4/10*
11.2 .+-. 4.7** 8.2 .+-. 0.1** (30 mg/kg/day) N-MCT 1-7 1/10 9/6
.+-. 2.6** 8.3 .+-. 0.2** (10 mg/kg/day) Cidofovir 1-2 9/10*** 8.0
7.0 .+-. 0.2** (100 mg/kg/day) Placebo 1-7 1/20 7.9 .+-. 1.8 8.7
.+-. 0.1 .sup.aTwice daily (12 h apart) for N-MCT and once daily
for cidofovir, starting 24 h after virus exposure. .sup.bMean day
of death .+-. SD of mice that died prior to day 21.
.sup.cLog.sup.10 PFU/g .+-. SD determined on day 5 of the
infection. *P < 0.5. **P < 0.01. ***P < 0.001.
[0174] The results of the experiments regarding activity of N-MCT
against a severe vaccinia (HID strain) respiratory infection in
mice (high virus challenge of 105 PFU/mouse) is presented in Table
7.
TABLE-US-00007 TABLE 7 Compound Treatment Survivors/ Mean Lung
(mg/kg/day).sup.a Days Total MDD.sup.b Virus Titer.sup.c N-MCT 1-7
10/10*** >21*** 7.1 .+-. 0.3** (100 mg/kg/day) N-MCT 1-7
10/10*** >21*** 8.1 .+-. 0.1** (30 mg/kg/day) N-MCT 1-7 10/10***
>21*** 8.3 .+-. 0.1** (10 mg/kg/day) Cidofovir 1-2 10/10***
>21*** 4.7 .+-. 0.6** (100 mg/kg/day) Placebo 1-7 4/20 8.2 .+-.
0.5 8.6 .+-. 0.3 .sup.aTwice daily (12 h apart) for N-MCT and once
daily for cidofovir, starting 24 h after virus exposure. .sup.bMean
day of death .+-. SD of mice that died prior to day 21.
.sup.cLog.sup.10 PFU/g .+-. SD determined on day 5 of the
infection. *P < 0.5. **P < 0.01. ***P < 0.001.
BALB/c Mice Inoculated with Cidofovir-Resistant Vaccinia WR
Strain
[0175] The effect of twice daily i.p. treatment with N-MCT
(compared to placebo or CDV) on the mortality of BALB/c mice
challenged with a cidofovir-resistant vaccinia WR strain was
investigated. N-MCT was prepared in 0.4% CMC and delivered i.p. in
0.1 ml doses to treat the resulting infection. CDV was prepared in
sterile saline and given i.p. in 0.1 ml doses. Animals were treated
twice daily for 7 days beginning 1 day after viral challenge.
Results of the testing (Table 8) indicated that N-MCT exhibited
greater efficacy against the vaccinia WR strain than CDV,
suggesting that N-MCT may be particularly well-suited for the
treatment of strains of poxvirus resistant to conventional
therapies.
TABLE-US-00008 TABLE 8 Treatment Number of Survivors Placebo -
Saline 0/20 CDV (100 mg/kg/day) 7/10 N-MCT (100 mg/kg/day)
10/10
Results
[0176] N-MCT fully protected the infected mice from death resulting
from infection by the vaccinia IHD strain at higher doses and was
still significantly protective at the lowest dose tested. Its
activity against cowpox was less dramatic compared to the case of
vaccinia infection; however, significant protection was still
observed at higher doses. In addition, the efficacy was
dose-responsive. When tested against a more virulent vaccinia WR
strain, N-MCT was not active. However, N-MCT was well tolerated by
the mice even at the highest concentration tested (100 mg/kg) and
N-MCT was active against a cidofovir (CDV)-resistant mutant strain,
indicating its viral target may be different from that of
cidofovir.
Experiment #2
Differences in Susceptibility of Pox Viruses to Antiviral Drugs
[0177] Both herpes simplex virus (HSV) and vaccinia virus (VV)
express thymidine kinase (TK) activity and each of these enzymes
can phosphorylate thymidine (see Kit, et al., 1967. J. Virol. 1,
238-240.; Kit, et al., 1963. J. Mol. Biol. 6, 22-33). These enzymes
differ in that the herpesvirus TK is a type I enzyme, whereas the
VV TK is a type II enzyme (Black, et al., 1992. J. Biol. Chem. 267,
9743-9748). The HSV type I TK is the product of the of the UL23
gene (McGeoch, et al., 1988. J. Gen. Virol. 69, 1531-1574), and
like all type I enzymes, is active as a homodimer and lacks
allosteric control (Jamieson, et al., 1974. J. Gen. Virol. 24,
481-492). This enzyme also exhibits a broad substrate specificity,
including thymidine, 2'-deoxycytidine and synthetic nucleoside
analogs (Coen, et al., 1980. Proc. Natl. Acad. Sci. U.S.A. 77,
2265-2269). VV TK is the prototypic type II enzyme and is encoded
by the J2R gene (Hruby, et al., 1983. Proc. Natl. Acad. Sci. U.S.A.
80, 3411-3415). Like other members of this class, it is active as a
homotetramer (Hruby, et al., 1982. J. Virol. 43, 403-409) and is
allosterically inhibited by both dTTP and dTDP (Black, et al.,
1992. J. Biol. Chem. 267, 6801-6806). The human TK is also a type
II enzyme and like its homolog in VV, exhibits a restricted
substrate specificity limited to thymidine and closely related
analogs.
[0178] It is hypothesized that the observed differences in
susceptibility to a number of antiviral drugs might be related to
the distinct classes of TK enzymes expressed in these virus
families. Thus, the poor activity of many nucleoside analogs
against cowpox virus (CV) may reflect their inability to be
activated by the type II TK expressed by this virus. To test this
hypothesis, the antiviral activity of selected compounds against
TK+ and TK- strains of both HSV-1 and CV was examined. Most of the
compounds examined did not exhibit HSV-1 TK dependence. This is
consistent with the notion that the poor phosphorylation of
nucleoside analogs contributes significantly to the lack of
activity for many compounds against orthopoxviruses. The approach
described here to investigate TK dependence in CV can also be used
to identify novel compounds that are preferentially activated by
the orthopoxvirus TK.
[0179] Cells and Viruses
[0180] Primary human foreskin fibroblast (HFF) cells were prepared
and passaged by methods described previously (Rybak, et al., 2000.
Antimicrob. Agents Chemother. 44, 1506-1511). Vero cells were
obtained from the American Type Culture Collection (Manassas, Va.).
The wild type (wt) HSV-1 strain F and TK.sup.- strain DM2.1 were
described and propagated as reported previously (Hartline, et al.,
2005. Antiviral Res. 65, 97-105). CV strains delta crmA (TK.sup.+)
and TK:GFP lacZ (TK.sup.-) were obtained from Pete Turner
(University of Florida, Gainesville, Fla.) and were described
previously (Ali, et al., 1994. Virology 202, 305-314). Cidofovir,
idoxuridine (IDU), 5-bromodeoxyuridine (BrdU), vidarabine (AraA),
5-fluorodeoxyuridine (FdU), trifluridine (TFT), fialuridine (FIAU),
fiacitabine (FIAC), sorivudine (BVAU), ACV and brivudin (BVDU) were
either purchased (Sigma-Aldrich, St. Louis, Mo.) or were obtained
through the NIAID, NIH, Bethesda, Md.
[0181] CV .beta.-Galactosidase Assay
[0182] Monolayers of Vero cells in 96-well plates were incubated at
37.degree. C. for 24 h in a humidified incubator. Drugs were then
diluted in the plates and either TK.sup.+ or TK.sup.- strains of CV
were added at a multiplicity of infection of 0.05 PFU/cell. At 72 h
post infection, the medium was removed and the .beta.-galactosidase
substrate, chlorophenol red-.alpha.-galactopyranoside, was added at
a final concentration of 50 .mu.g/ml in a phosphate-buffered saline
solution. The conversion of the colorimetric substrate was
determined by measuring the absorbance at 570 nm, and 50% effective
concentration (EC.sub.50) values were calculated. The
TK.sup.-/TK.sup.+ EC.sub.50 ratio was calculated in each experiment
and an average ratio and standard deviation were calculated for all
experiments.
[0183] HSV-1 Plague Reduction Assay
[0184] HFF cells were seeded into six-well plates and incubated at
37.degree. C. Two days later, drug was serially diluted 1:5 in MEM
with 2% FBS using six concentrations of drug with a starting
concentration of 100 .mu.g/ml. Viruses were diluted in MEM
containing 10% FBS to a concentration that yielded 20-30 plaques
per well. The media were then aspirated from the wells and 0.2 ml
of virus was added to each of triplicate wells with 0.2 ml of
medium being added to control wells. The plates were then incubated
for 1 h with shaking every 15 min and drug was added to appropriate
wells. After an incubation period of 3 days, the cells were stained
with 0.1% crystal violet in 20% MeOH. The stain was aspirated, the
wells washed with PBS and the plaques enumerated using a
stereomicroscope. EC.sub.50 values were calculated in a standard
manner.
[0185] Cytotoxicity Assay
[0186] To determine the toxicity of drugs, HFF cells were seeded
into 96-well plates at a concentration of 2.5.times.10.sup.4
cells/well in growth media. After 24 h, the media were aspirated
and 100 .mu.g/ml of drug was added to the first row of the plate
and five-fold serial dilutions were performed. Following a 7 day
incubation, the media were aspirated and 660 .mu.g/ml of neutral
red stain was added and incubated for 1 h. The monolayers were then
washed and the dye was dissolved in a solution containing 50%
ethanol and 1% glacial acetic acid. The plates were mixed for 15
min on a rotating shaker and the optical densities were determined
at 550 nm. CC.sub.50 values were interpolated from the data.
[0187] Genetic Resistance Assays
[0188] The principle behind both the CV and HSV-1 genetic
resistance assays is the same. The efficacy of antiviral drugs was
determined in TK.sup.+ and TK.sup.- strains of the same virus and
the ratio of the EC.sub.50 against the TK.sup.- virus to the
EC.sub.50 against the TK.sup.- virus was used as a measure of TK
dependence. The nucleotide analog cidofovir (CDV) was used as a
negative control for both these viruses. As positive controls,
acyclovir (ACV) and 5-iododeoxyuridine (IdU) were used in HSV-1 and
CV assays, respectively, since these drugs require phosphorylation
for their antiviral activity.
[0189] Amino Acid Alignment and Phylogeny
[0190] Amino acid sequences for TK homologs were downloaded from
GenBank and subjected to a clustal W alignment and unrooted
phylogenetic trees were constructed using Vector NTI (Invitrogen,
San Diego, Calif.). Gene ID numbers in the analysis and
abbreviations used are VV, 29692200; HSV-1, 9629403; HSV-2,
9629292; Homo sapiens (human), 4507519; goatpox virus, 55274605;
VZV, 118822; EBV, 23893647; variola virus, 66679; fowlpox virus,
221413; monkeypox virus, 22096356; CV, 20178469.
[0191] Results
[0192] A set of antiviral drugs was selected based on previously
described activity against HSV-1 and orthopoxviruses. Since IDU has
been used in the laboratory to select for TK-deficient
orthopoxviruses (Byrd, et al., 2004. Methods Mol. Biol. 269, 31-40)
and a number of analogs have good activity against VV (De Clercq,
E., 1980. Methods Find. Exp. Clin. Pharmacol. 2, 253-267),
additional analogs were studied to determine if this gene was
involved in the activation of other compounds in this series. The
structures of these compounds were as follows:
##STR00017## ##STR00018##
[0193] A total of 11 compounds including control drugs were tested
in the genetic resistance assays and the EC.sub.50 ratios were
calculated. Results for antiviral activity of the compounds against
TK.sup.+.sub.endTK.sup.- strains of HSV-1 and CV are presented in
Table 9.
TABLE-US-00009 TABLE 9 HSV-1 CV F.sup.a EC.sub.50 DM2.1.sup.a
EC.sub.50 delta crmA.sup.c TK:GFP.sup.c EC.sub.50 Toxicity.sup.d
(TK+) EC.sub.50 (TK-) ratio.sup.b EC.sub.50 (TK.sup.+) lacZ
EC.sub.50 (TK.sup.-) ratio.sup.b CC.sub.50 ACV 0.35 .+-. 0.2
>100 .+-. 0 >286 >30 >30 1 >100 .+-. 0 CDV 1.5 .+-.
1.1 1.5 .+-. 1.4 1 16.1 .+-. 0.30 3.3 .+-. 2.3 0.54 >100 .+-. 0
IDU 2.1 .+-. 0.1 54 .+-. 8.1 26 2.2 .+-. 0.87 16 .+-. 9.3 7.3
>100 .+-. 0 AraA 6.4 .+-. 3.4 4.9 .+-. 0 0.78 0.91 .+-. .005
0.78 .+-. 0.2 0.86 >100 .+-. 0 BrdU 1.6 .+-. 0.2 39 .+-. 6.3 24
0.37a .+-. 0 19a .+-. 15 51 >100 .+-. 0 FdU 2.5 .+-. 1.7 4.5
.+-. 3.3 1.8 0.37a .+-. 0 0.37a .+-. 0 1 60 .+-. 0.7 TFT 1.0 .+-.
0.2 1.1 .+-. 0.3 1.1 0.42 .+-. 0.33 0.72 .+-. 0.28 1.7 >100 .+-.
0 FIAU 0.05 .+-. 0.03 3.9 .+-. 3.3 78 14 .+-. 7.0 19 .+-. 6.7 1.4
>100 .+-. 0 FIAC 0.06 .+-. 0.02 8.0 .+-. 1.3 133 18 .+-. 0.25
9.2 .+-. 3.7 0.51 >100 .+-. 0 BVDU 0.09 .+-. 0 100 .+-. 0 1111
>30 >30 1 >100 .+-. 0 BVAU 0.05 .+-. 0.02 >100 .+-. 0
>2000 >30 >30 1 >100 .+-. 0 .sup.aAverage of two
experiments .+-. standard deviation (.mu.g/ml). .sup.bRatio of
EC.sub.50 values for TK.sup.- and TK.sup.+ strains of the virus.
.sup.cAverage of five experiments .+-. standard deviation.
.sup.dDetermined by duplicate neutral red uptake assays.
[0194] For HSV-1, the wt strain was very sensitive to ACV, while
the TK.sup.- strain was highly resistant to the drug and yielded an
EC.sub.50 ratio of 286. In contrast, EC.sub.50 values for the CDV
negative control were indistinguishable between these two strains
yielding an EC.sub.50 ratio of 1, confirming that the assay could
identify compounds that require TK in their mechanism of action.
TFT, FdU and AraA did not appear to be selectively phosphorylated
to a significant extent by the HSV-1 TK since the EC.sub.50 ratios
were very close to 1, but each of the remaining compounds was
significantly less effective against the TK.sup.- virus suggesting
not only that this enzyme was required for their activation. Among
these, ACV, BVAU, BVDU, FIAC, and FIAU appeared to be the most
dependent on this enzyme suggesting that they are not
phosphorylated to a significant extent by host kinases. IDU and
BrDU were dependent on TK, but retained some activity in the
TK.sup.- virus suggesting that they might be activated to some
degree by enzymes other than the HSV-1 TK (type 1).
[0195] This same set of compounds was also tested against CV, and
EC.sub.50 ratios were calculated to assess the ability of the type
II TK from this virus to activate these same compounds (Table 9).
Both strains of CV were fully sensitive to the CDV control and
yielded an EC.sub.50 ratio of 0.54. The TK.sup.+ strain of CV was
sensitive to IDU as reported previously for CV (Kern, E. R., 2003.
Antiviral Res. 57, 35-40; Smee, et al., 2004. Nucleosides,
Nucleotides Nucleic Acids 23, 375383) and vaccinia virus (Kern, E.
R., 2003. Antiviral Res. 57, 35-40; Neyts, et al., 2002.
Antimicrob. Agents Chemother. 46, 2842-2847), while the TK.sup.- CV
strain was significantly less sensitive to the drug and yielded an
EC.sub.50 ratio of 7.3. These results indicated that the assay was
capable of identifying compounds that require TK for their
activity. The closely related analog, BrdU, was also much less
effective against the TK.sup.- virus and yielded a
TK.sup.-/TK.sup.+ EC.sub.50 ratio of >50. Both TFT and FdU were
equally effective in inhibiting both strains of CV and HSV-1, as
was expected since both these molecules are inhibitors of
thymidylate synthetase (Emura, et al., 2004. Int. J. Mol. Med. 13,
249-255). The antiviral activity of AraA was also independent of TK
and is consistent with results obtained with HSV-1. ACV, BVDU, and
BVAU did not significantly inhibit replication of either CV strain,
suggesting that either they are not activated by the CV-encoded TK
or they are not substrates for the viral DNA polymerase. Both FIAC
and FIAU exhibited modest activity against TK.sup.- and TK.sup.+
strains of the virus suggesting that the compounds can inhibit the
CV DNA polymerase, but also that the TK expressed by CV does not
appear to activate the compounds to a measurable extent. This
contrasts with the high EC.sub.50 ratios for FIAC and FIAU in HSV-1
of 78 and 133, respectively.
[0196] To confirm results from the TK dependence studies, enzyme
kinetics were determined for N-MCT and other substrates using
purified VV TK in enzymatic assays. Enzyme kinetic parameters for
substrates of VV TK are provided in Table 10. Several thymidine
analogs, including N-MCT had Kin values that were comparable to
thymidine suggesting that they were good substrates for the enzyme.
Values for V.sub.max were also obtained and were used to calculate
the efficiency (V.sub.max/K.sub.m) for each of the substrates.
These results suggested that N-MCT was efficiently phosphorylated
by the enzyme.
TABLE-US-00010 TABLE 10 V.sub.max Substrate K.sub.m (.mu.M).sup.a
(.mu.M min.sup.-1 mg.sup.-1) V.sub.max/K.sub.m dThd 49 .+-. 7.6 289
.+-. 137 5.9 BrdU 30 .+-. 6.2 281 .+-. 59 9.4 IdU 31 .+-. 4.7 222
.+-. 58 7.2 FdU 113 .+-. 85 108 .+-. 63 0.96 TFT 14 .+-. 4.3 333
.+-. 44 24 FIAU 4.3 .+-. 2.5 99 .+-. 41 22.7 N-MCT 32 .+-. 14 114
.+-. 23 3.6 FIAC >135 <0.14 <0.001 CDV >158 <0.025
<0.0002 .sup.aAverage of four or more determinations with
standard deviations as shown.
[0197] The effect of HSV-1 TK and CV TK on the antiviral activity
of several related nucleoside analogs was tested. In these assays,
a positive result is significant in that it suggests that TK is
involved in the mechanism of action of the drug, and also implies
selectivity since viral TK gene must be dominant over cellular
kinases. It does not demonstrate the phosphorylation of the drugs
directly, but suggests that it may be involved given the kinase
activity of the enzymes. There are many potential explanations for
negative results including the possibility that both cellular and
viral enzymes could be activating the drug. Closely related
nucleoside analogs were selected because the orthopoxviruses
express type II TK enzymes and were expected to have a narrow
substrate specificity.
[0198] Results presented were consistent with this prediction since
very few analogs of IDU appear to be activated by CV TK. The narrow
substrate specificity of this enzyme was apparent even with the
small set of compounds tested. As expected, IDU was dependent on
the CV TK and the substitution of bromine for iodine was well
tolerated by the enzyme as evidenced by the high TIC/TK+EC.sub.50
ratio of 51. However, the substitution of a larger bromovinyl group
at this same position eliminated its activity against both strains
of CV and this is consistent with a lack of activation by CV TK,
but it is also possible that the triphosphate metabolite of this
compound did not inhibit the CV DNA polymerase.
[0199] The introduction of either fluorine or a trifluoromethyl
group at the C-5 position makes FdU and TFT inhibitors of
thymidylate synthetase and predictably, both these molecules
inhibit TK.sup.+ and TK.sup.- strains of both HSV-1 and CV.
Although TFT does not exhibit TK dependence in HSV, the viral
enzyme can phosphorylate the drug to a limited extent and the
monophosphorylated drug can be detected in TK cells (Field, et al.,
1981. J. Infect. Dis. 143, 281-285).
[0200] Substitutions on the sugar moiety also appear to be poorly
tolerated since the addition of a fluorine at the 2' position in
FIAU rendered it equally effective against both strains of CV.
Results from FIAC and FIAU were particularly informative since both
these drugs exhibit modest antiviral activity against CV, and this
is consistent with the compounds inhibiting the DNA polymerase.
However, neither of these compounds appeared to be activated by the
type II TK encoded by this virus and contrasts with results in
HSV-1, where both these compounds were efficiently phosphorylated
by the type I TK encoded by this herpesvirus. The fact that the
relative potency of these compounds against CV is similar to that
against the TK.sup.- strain of HSV-1 is also consistent with this
interpretation of the data.
[0201] The results obtained confirm that the CV TK has a rather
limited substrate specificity compared to the TK encoded by HSV-1.
This outcome was expected since this virus encodes a type II TK
homolog that is more closely related to the human enzyme than the
enzyme from HSV-1 (FIG. 1). TK homologs encoded by UV, CV, variola
virus and monkeypox virus are all quite similar compared to the
high degree of sequence divergence in the type I enzymes encoded by
the herpesviruses (FIG. 2). The high degree of similarity among the
human orthopoxviruses TK homologs is important and suggests that
the assay reported will be predictive for variola virus and
monkeypox virus. The high degree of amino acid identity among the
orthopoxvirus enzymes and the human homolog as well as the narrow
substrate specificity that is characteristic of type II enzymes
will likely make it difficult to identify nucleoside analogs that
are only activated by these enzymes. Nevertheless, it should be
possible to identify nucleosides that are selectively
phosphorylated by the orthopoxvirus TK homolog (FIG. 2). Once
activated in cells infected with CV, cellular enzymes could
phosphorylate the compounds to the level of the triphosphate to
make them inhibitors of the viral DNA polymerase. The CV genetic
resistance assay described herein is capable of identifying
inhibitors that depend on the viral TK for their activity.
[0202] TDNA polymerase inhibitors were developed that are
specifically activated by the type I TK molecules encoded by the
herpesviruses, and antiviral drugs with this same mechanism of
actions can be developed against orthopoxviruses. Specificity
derived through selective phosphorylation is a highly effective
strategy for the treatment of orthopoxvirus infections, and
compounds that act by such a mechanism can be developed by
identifying drugs that require the viral TK for their activity.
Compounds thus identified are expected to have a relatively good
toxicity profile since, if they remain unphosphorylated, they are
unlikely to inhibit cellular polymerases, and thus will be useful
new therapies for the treatment of orthopoxvirus infections.
Experiment #3
Antiviral Activity of N-MCT Against Orthopoxviruses
[0203] The antiviral activity of N-MCT against the orthopoxviruses
was confirmed and the function of TK in the mechanism of action of
this compound was investigated. These studies suggested that this
molecule was specifically phosphorylated by the TK homologs in both
CV and HSV-1 and that the active metabolite inhibited viral
replication at the level of viral DNA synthesis. These data taken
together with the pharmacokinetic properties of the drug (Noy, et
al. 2002. Cancer Chemother. Pharmacol. 50:360-366) suggested that
it might exhibit antiviral activity in vivo. N-MCT was tested in a
murine model against UV, CV, and HSV-1. Mice treated
intraperitoneally (i.p.) with N-MCT beginning 24 h after infection
were protected from lethality with each of these viruses at
relatively low doses of drug. N-MCT is a molecule that can be
activated by the divergent TK homologs in orthopoxviruses and some
herpesviruses and thus can be useful in therapy for these
infections in humans.
[0204] Cells and Viruses
[0205] Methods for producing and passaging human foreskin
fibroblast (HFF) cells were described previously (Rybak, et al.
2000. Antimicrob. Agents Chemother. 44:1506-1511). Culture medium
for all cell lines was minimal essential medium (MEM) containing
10% fetal bovine serum (FBS) and standard concentrations of
L-glutamine, penicillin, and gentamicin. VV strains WR, Copenhagen,
and IHD were obtained from the American Type Culture Collection
(ATCC; Manassas, Va.). Working stocks of these viruses were
propagated in Vero cells obtained from the ATCC. CV, strain
Brighton, was provided by John W. Huggins (Department of Viral
Therapeutics, Virology Division, U.S. Army Medical Research
Institute of Infectious Disease, Frederick, Md.). .DELTA.crmA
(TK.sup.+) and TK:GFP lacZ (TK.sup.-) CV strains were obtained from
Pete Turner (University of Florida, Gainesville, Fla.) and were
described previously (Ali, et al. 1994. Virology 202:305-314). The
wild-type HSV-1 strain F and TK.sup.- strain DM2.1 were described
and propagated as reported previously (Hartline, et al. 2005.
Antivir. Res. 65:97-105). CDV (Vistide) was provided by Gilead
Pharmaceuticals (Foster City, Calif.), and N-MCT and the other
compounds were obtained through the NIAID, NIH, Bethesda, Md.
[0206] VV, CV, and HSV Plaque Reduction Assays
[0207] For VV and CV, HFF cells were added to six-well plates and
incubated for 2 days at 37.degree. C. with 5% CO.sub.2 and 90%
humidity. On the day of assay, drug at two times the final desired
concentration was diluted serially 1:5 in 2.times.MEM with 10% FBS
to provide six concentrations. Aspiration of culture medium from
triplicate wells for each drug concentration was followed by
addition of 0.2 ml per well of diluted virus, which would give 20
to 30 plaques per well in MEM containing 10% FBS or 0.2 ml medium
for drug toxicity wells. The plates were incubated for 1 h with
shaking every 15 min. An equal amount of 1% agarose was added to an
equal volume of each drug dilution, and this mixture was added to
each well in 2 ml volumes and the plates were incubated for 3 days.
The cells were stained with a solution of neutral red in
phosphate-buffered saline (PBS) and incubated for 5 to 6 h. The
stain was aspirated, plaques were counted using a stereomicroscope
at 10.times. magnification, and 50% effective concentrations
(EC.sub.50s) were calculated by standard methods. The HSV plaque
reductions were essentially the same as those for VV and CV with
the following changes. The drug solutions were prepared at the
desired concentration in MEM with 2% FBS, and a liquid overlay with
pooled human serum containing antibodies to HSV instead of agarose
was used. At 72 h following infection, the medium containing the
drug was aspirated and the monolayers were stained with 1 ml of a
solution of 0.01% crystal violet in 60% methanol for 10 min.
Residual stain was then washed from the wells with 1 ml PBS, and
plaques were counted.
[0208] CV .beta.-Galactosidase Assay
[0209] Monolayers of HFF cells in 96-well plates were incubated at
37.degree. C. for 24 h in a humidified incubator. Drugs were then
diluted in the plates, and either TK+ or TK strains of CV were
added at a multiplicity of infection of 0.05 PFU/cell (Prichard, et
al. Distinct thymidine kinases encoded by cowpox virus and herpes
simplex virus contribute significantly to the differential
antiviral activity of nucleoside analogs. Antivir. Res., in press).
At 48 h postinfection, the medium was removed and the
.beta.-galactosidase substrate chlorophenol
red-.beta.-galactopyranoside was added at a final concentration of
50 .mu.g/ml in PBS. The conversion of the calorimetric substrate
was determined by measuring the absorbance at 570 rum, and
EC.sub.50s were calculated by standard methods (Prichard, et al.
1990. Antivir. Res. 14:181-205). The EC.sub.50 ratio for TK+ and TK
viruses was calculated and used as a measure of TK dependence.
[0210] Cytotoxicity Determination
[0211] The neutral red uptake assay was conducted as reported
previously (Keith, et al. 2003. Antimicrob. Agents Chemother.
47:2193-2198). Briefly, HFF cells were plated into 96-well plates
at a concentration of 2.5.times.10.sup.4 cells per well. After 24
h, the medium was aspirated and 125 .mu.l of each drug
concentration in MEM with 2% FBS was added to the first row of
wells in triplicate. Serial 1:5 dilutions were performed using the
Beckman BioMek liquid handling system. After compound addition, the
plates were incubated for 7 days in a CO.sub.2 incubator at
37.degree. C. After incubation, the medium/drug was aspirated and
200 .mu.l/well of 0.01% neutral red in PBS was added and incubated
for 1 h. The dye was aspirated, and the cells were washed with PBS
using a Nunc plate washer. After the PBS was removed, 200
.mu.l/well of a solution containing 50% ethanol and 1% glacial
acetic acid was added. The plates were placed on a rotary shaker
for 15 min, and the optical densities were determined at 540 nm.
The concentration of drug that reduced cell viability by 50% was
then calculated. In the proliferation assay, cells were seeded in
six-well plates at a concentration of 2.5.times.10.sup.4 cells/ml.
After 24 h, the medium was aspirated and drug serially diluted 1:5
was added to the appropriate wells. The cells were incubated for 72
h at 37.degree. C. and then trypsinized and counted using a Coulter
counter. Standard methods were used to determine the drug
concentration which inhibited cell proliferation by 50%.
[0212] DNA Synthesis Assay
[0213] Drugs were diluted to final concentrations of 30, 10, 3, 1,
and 0.3 .mu.g/ml and added to confluent monolayers of HFF cells in
six-well plates. The WR strain of W was used to infect the cells at
a multiplicity of infection of 0.5 PFU/cell. Total DNA was
extracted 24 h postinfection by lysing the cells in a buffer
containing 10 mM Tris, pH 7.4, 1 mM EDTA, and 1% sodium dodecyl
sulfate and incubating them with 100 .mu.g of proteinase K for 60
min at 37.degree. C. DNA samples were purified using QIAGEN's
QiaQuick PCR purification system according to the manufacturer's
protocol. Samples were cleaved with EcoRV, and fragments were
separated on an agarose gel, transferred to a nylon membrane, and
hybridized to a digoxigenin-labeled probe (Roche Applied Science,
Indianapolis, Ind.), specific for W DNA (coordinates 128257 to
129537 in AY243312).
[0214] Activity of N-MCT in Mice Inoculated with HSV, VV, or
CV.
[0215] Female BALB/c mice, 3 to 4 weeks of age, were obtained from
Charles River Laboratories (Raleigh, N.C.). Mice were group housed
in microisolator cages, and each treatment group contained 15 mice.
Mice were obtained, housed, utilized, and euthanized according to
policies of the USDA and AAALAC. All animal procedures were
approved by the University of Alabama at Birmingham Institutional
Animal Care and Use Committee prior to initiation of studies.
Infections were initiated by intranasal (i.n.) inoculation of
BALB/c mice. Mice were infected with an approximate 90% lethal dose
of HSV-1 strain E377 (1.0.times.10.sup.5 PFU/animal), CV strain BR
(3.3.times.10.sup.4 PFU/animal), VV strain WR (1.0.times.10.sup.4
PFU/animal), or VV strain IHD (2.5.times.10.sup.4 PFU/animal) using
a micropipettor and a total volume of 40 .mu.l per animal. N-MCT
was suspended in 0.4% carboxylmethyl cellulose (CMC) to yield 50-,
16.7-, or 5.6-mg/kg-of-body-weight doses in a volume of 0.1 ml.
Mice were treated i.p. with N-MCT twice daily, approximately 12 h
apart beginning 24 h post-viral inoculation with a 5-day duration
of therapy for orthopoxviruses and a 7-day duration for HSV-1.
Vehicle-treated mice were included as negative controls, and either
ACV or CDV was included as a positive control. Mortality rates were
analyzed by Fisher's exact test, and mean day of death was analyzed
by Mann-Whitney U rank sum. A P value of 0.05 or less was
considered significant.
[0216] Results
[0217] The antiviral activity of N-MCT was characterized and N-MCT
was evaluated for its potential as a therapy for orthopoxvirus and
HSV infections. Efficacy and cytotoxicity of N-MCT was determined
in HFF cells infected with the Copenhagen strain of VV and the
Brighton strain of CV (Table 11).
TABLE-US-00011 TABLE 11 VV CV (Copenhagen) (Brighton) Drug
EC.sub.50.sup.c SI.sup.d EC.sub.50.sup.c SI.sup.d CC.sub.50.sup.a
IC.sub.50.sup.b N-MCT 0.55 .+-. 0.13 >182 1.5 .+-. 1.2 >67
>100 .+-. 0 29 .+-. 5.4 CDV 3.3 .+-. 0.3 >30 4.4 .+-. 0.6
>23 >100 .+-. 0 20 .+-. 7 .sup.aCC.sub.50, 50% cellular
cytotoxicity determined by neutral red uptake in stationary
monolayers of HFF cells. .sup.bIC.sub.50, 50% inhibition of cell
proliferation. .sup.cAverage of two or more assays shown in units
of .mu.g/ml with standard deviations. .sup.dSI, selective index
calculated as the CC.sub.50 divided by the EC.sub.50.
[0218] In these standard plaque assays, the drug yielded EC.sub.50s
against both viruses that were lower than those for the CDV
control. Cytotoxicity was also determined in both a neutral red
uptake assay and a proliferation assay and appeared to be at least
comparable to that of CDV, resulting in selective indices that were
higher than those for CDV.
[0219] Previous reports suggested that N-MCT was a substrate of the
HSV-1 TK, since an inhibitor of this enzyme reduced the
phosphorylation of the drug (Zalah, et al 2002. Antivir. Res.
55:63-75), and the enzyme supplied in trans conferred sensitivity
to the drug in uninfected cells (Schelling, et al. 2004. J. Biol.
Chem. 279:32832-32838). It is hypothesized that the type II TK
homologs expressed by orthopoxviruses might also be capable of
activating this compound, since its structure was very close to the
natural substrate of the enzyme. The antiviral activity of N-MCT
against TK.sup.+ and TK.sup.- strains of CV and HSV-1 in a genetic
resistance assay was determined (Prichard, et al. Distinct
thymidine kinases encoded by cowpox virus and herpes simplex virus
contribute significantly to the differential antiviral activity of
nucleoside analogs. Antivir. Res., in press). Drugs that require TK
for their activation exhibit elevated TK.sup.-/TK.sup.+ EC.sub.50
ratios, whereas compounds that do not require the enzymes to be
active yield EC.sub.50 ratios near unity. In HSV-1, ACV was used as
a positive control since it required phosphorylation by the TK and
CDV was used as a negative control since it does not require
phosphorylation by the enzyme to be active. The high EC.sub.50
ratios for ACV and idoxuridine (IDU) reflect the fact that they
require phosphorylation by this enzyme (Table 12).
TABLE-US-00012 TABLE 12 EC.sub.50 (.mu.g/ml) CV CV HSV-1
(.DELTA.crm lacZ) (TK:gpf lacZ) HSV-1 (DM2.1) Drug TK.sup.+
TK.sup.+ Ratio.sup.b (F) TK.sup.+ TK.sup.- Ratio.sup.b N-MCT 2.0
.+-. 2.0a 28 .+-. 2.3 14 0.07 .+-. 0.02 6.3 .+-. 3.9 90 CDV 3.5
.+-. 3.3 4.2 .+-. 3.9 1.2 1.5 .+-. 1.1 1.5 .+-. 1.4 1.0 ACV
NA.sup.c NA NA 0.3 .+-. 0 >100 .+-. 0 333 IDU 0.4 .+-. 0.18 22
.+-. 9.3 55 2.1 .+-. 0.1 54 .+-. 8.1 26 aAverage of three or more
experiments with standard deviation. .sup.bEC.sub.50 ratio for
TK.sup.- and TK.sup.+ viruses, respectively. .sup.cNA, not
applicable.
[0220] N-MCT also yielded an elevated EC.sub.50 ratio, confirming
that it was phosphorylated by TK, while the CDV negative control
was unaffected by the deletion of this gene. In CV, IDU was used as
a positive control since it was known to be activated by its TK,
and CDV was used as a negative control. CDV was equally effective
against the two viruses and yielded an EC.sub.50 ratio of 1.2,
while IDU was much less effective against the TK.sup.- strain of CV
and yielded an EC.sub.50 ratio of 55 (Table 12). The TK virus was
also significantly less sensitive to N-MCT and yielded an EC.sub.50
ratio of 14, suggesting that this compound may also require TK for
its activation. The ability of this compound to inhibit DNA
synthesis was determined using CDV as a control. N-MCT was at least
as effective in inhibiting VV DNA synthesis as the CDV positive
control and yielded EC.sub.50s of 1 and 3 .mu.g/ml, respectively
(FIG. 3). Both of these values were comparable to the antiviral
EC.sub.50s of 0.6 and 3 .mu.g/ml, respectively, and were sufficient
to explain the antiviral effects of the drug. The antiviral
activity of the compound in vitro taken together with its
pharmacokinetic parameters (Noy, et al. 2002. Cancer Chemother.
Pharmacol. 50:360-366) and the mechanism of action studies suggest
that it should be an effective and highly selective compound in
vivo. An established animal model was used to evaluate the
antiviral activity of the compound against HSV-1 (Kern, et al.
1973. J. Infect. Dis. 128:290-299). BALB/c mice were infected i.n.
with the E377 strain of HSV-1, and animals were treated twice daily
beginning 24 h after infection. In this experiment, 7 of 14 animals
succumbed to the infection, suggesting that the inoculum was
slightly lower than the 90% lethal dose target dose. Nevertheless,
all doses of the drug protected mice from mortality (P<0.01) and
were comparable to the ACV positive control (Table 13).
TABLE-US-00013 TABLE 13 Mortality Treatment.sup.a No. dead/total
no. % P value MDD.sup.b P value Placebo 0.4% CMC 7/14 50 8.0 ACV 50
mg/kg 0/15 0 <0.01 16.7 mg/kg 0/15 0 <0.01 5.6 mg/kg 0/15 0
<0.01 N-MCT 50 mg/kg 0/15 0 <0.01 NS.sup.c 16.7 mg/kg 0/15 0
<0.01 NS 5.6 mg/kg 1/15 7 <0.05 20 0.05 .sup.aN-MCT was
prepared in 0.4% CMC and delivered i.p. twice daily in 0.1-ml
doses. ACV was prepared in sterile water and given i.p. twice daily
in 0.1 ml doses. All animals were treated for 7 days beginning 24 h
postinfection. .sup.bMDD, mean day of death. .sup.cNS, not
significant.
[0221] These data suggest that this compound can be useful in the
treatment of HSV infections. Established models of orthopoxvirus
infections were also used to evaluate the efficacy of this compound
against lethal VV and CV infections in BALB/c mice (Quenelle, et
al. 2004. Antimicrob. Agents Chemother. 48:404-412). In the first
study, groups of 15 animals were infected i.n. with a lethal dose
of the IHD strain of VV. Drug was administered i.p. twice daily at
doses of 50, 16.7, and 5.6 mg/kg starting 24 h post-i.n.
inoculation. Mortality in all mice treated with vehicle was 100%,
whereas all mice treated with CDV at 15 mg/kg given once daily
survived (Table 14).
TABLE-US-00014 TABLE 14 Strain and treatment.sup.a Mortality
(mg/kg) No. dead/total no. % P value MDD.sup.b P value VV strain
IHD 0.4% CMC 15/15 100 7.6 CDV 15 0/15 0 <0.001 N-MCT 50 0/15 0
<0.001 16.7 0/15 0 <0.001 5.6 3/15 20 <0.001 8.7 <0.05
VV strain WR 0.4% CMC 15/15 100 7.6 CDV 15 0/15 0 <0.001 N-MCT
50 0/15 0 <0.001 16.7 2/15 13 <0.001 7.5 NS.sup.c 5.6 12/15
80 NS 8.2 NS CV strain Brighton 0.4% CMC 15/15 100 7.6 CDV 15 0/15
0 <0.001 N-MCT 50 2/15 13 <0.001 7.5 NS 16.7 3/15 20
<0.001 13.3 0.01 5.6 6/15 40 <0.001 14.0 0.05 .sup.aN-MCT was
prepared in 0.4% carboxymethyl cellulose and delivered i.p. twice
daily in 0.1-ml doses. CDV was prepared in sterile saline and given
i.p. once daily in 0.1-ml doses. All animals were treated for 5
days beginning 24 h postinfection. .sup.bMDD, mean day of death.
.sup.cNS, not significant.
[0222] All animals treated with 50 or 16.7 mg/kg N-MCT administered
twice daily survived, and 80% survived at the 5.6 mg/kg dose. All
treatments resulted in significantly improved mortality compared to
that of vehicle treated controls (P<0.001). The successful
treatment of the IHD strain of VV prompted us to repeat the
experiment using the WR strain, which is a more pathogenic strain
in our experimental model and has also been reported to be more
lethal by others (Smee, et al. 2004. Int. J. Antimicrob. Agents
23:430-437). All mice survived when treated with CDV at 15 mg/kg,
and mortality was 100% in vehicle-treated control mice (Table 14).
All animals treated with N-MCT at a dose of 50 mg/kg survived the
infection, and mortality was 13% and 80% at the 16.7 and 5.6 mg/kg
dose regimens given twice daily, respectively. In this experiment
the drug significantly protected mice at the high and medium dose
(P<0.001) but there was no significant protection at the low
dose. These data are consistent with the first experiment and also
with the notion that the WR strain is a slightly more virulent
virus in this model. The same experiment was repeated against the
Brighton strain of CV to confirm that N-MCT was active against
another orthopoxvirus. In this experiment, the CDV control
protected all animals from lethal infection with CV and all
vehicle-treated animals died (Table 14). Animals treated at all
doses of the test drug were significantly protected from mortality
(P<0.001), although a few animals died in each of the three
treatment groups. These data taken together indicate that N-MCT is
highly effective in treating orthopoxvirus infections in vivo.
[0223] Antiviral activity of N-MCT against HSV-1, VV, and CV both
in vitro and in vivo was demonstrated. This compound is unusual in
that it exhibits TK dependence both in a herpesvirus and in an
orthopoxvirus, and its rather uncommon spectrum of activity appears
to reflect its selective phosphorylation in cells infected with
these viruses. While many nucleoside analogs can be activated by
the type I TK homologs encoded by some of the herpesviruses, few
are apparently phosphorylated by the distinct type II TK molecules
expressed by orthopoxviruses and including IDU, bromodeoxyuridine
(De Clercq, E. 2001. Clin. Microbiol. Rev. 14:382-397), and N-MCT
as described herein. A previous report suggested that the
metabolism of this compound required the enzymatic activity of the
HSV-1 TK and that the compound was converted to N-MCT triphosphate,
which was incorporated in cellular DNA by the host polymerase
(Marquez, et al. 2004. J. Am. Chem. Soc. 126:543-549). The data
suggest that the compound may inhibit both orthopoxvirus and
herpesvirus DNA polymerases and impair viral DNA synthesis by a
similar mechanism. Alternative interpretations of the data exist,
and it is possible that N-MCT interferes with orthopoxvirus DNA
synthesis indirectly through the inhibition of thymidylate
synthetase by the monophosphate form of the drug. It is also
possible, albeit unlikely, that differences in drug activities
against CV are caused by genetic differences outside the TK locus.
Although additional experiments will be required to confirm
directly the inhibition of the VV DNA polymerase by N-MCT
triphosphate, the active site is highly conserved between this
enzyme and its homolog in herpes simplex virus. This proposed
mechanism of action appears to be sufficient to explain the
spectrum of activity of this compound. It is active against the
orthopoxviruses and the alphaherpesviruses since they express
enzymes with TK activity. The gammaherpesvirus Epstein-Barr virus
also encodes a TK homolog, and the drug inhibits its replication
with an EC.sub.50 of 0.45 .mu.g/ml (data not shown). Human
herpesvirus 8 also expresses a TK homolog and also appears to be
sensitive to the drug (Zhu, et al. 2005. Antimicrob. Agents
Chemother. 49:4965-4973). In contrast, this compound appears to be
inactive against the betaherpesviruses (human cytomegalovirus and
human herpesvirus 6) which do not express an enzyme with TK
activity, an observation which is also consistent with this mode of
action. The property of TK dependence is significant since it
should confer the desirable characteristics of good antiviral
activity and minimal toxicity in vivo. N-MCT is a compound with an
interesting spectrum of activity that is conferred by its selective
phosphorylation by some herpesvirus and orthopoxvirus TK homologs,
and can be used to treat both herpesvirus and orthopoxvirus
infections.
[0224] Methods and compositions that are suitable for use in
conjunction with aspects of the preferred embodiments are disclosed
in U.S. Pat. No. 5,840,728; U.S. Pat. No. 5,629,454; and U.S. Pat.
No. 5,869,666.
[0225] All references cited herein, including but not limited to
published and unpublished applications, patents, and literature
references are incorporated herein by reference in their entirety
and are hereby made a part of this specification. To the extent
publications and patents or patent applications incorporated by
reference contradict the disclosure contained in the specification,
the specification is intended to supersede and/or take precedence
over any such contradictory material.
[0226] The term "comprising" as used herein is synonymous with
"including," "containing," or "characterized by," and is inclusive
or open-ended and does not exclude additional, unrecited elements
or method steps.
[0227] All numbers expressing quantities of ingredients, reaction
conditions, and so forth used in the specification are to be
understood as being modified in all instances by the term "about."
Accordingly, unless indicated to the contrary, the numerical
parameters set forth herein are approximations that may vary
depending upon the desired properties sought to be obtained. At the
very least, and not as an attempt to limit the application of the
doctrine of equivalents to the scope of any claims in any
application claiming priority to the present application, each
numerical parameter should be construed in light of the number of
significant digits and ordinary rounding approaches.
[0228] The above description discloses several methods and
materials of the present invention. This invention is susceptible
to modifications in the methods and materials, as well as
alterations in the fabrication methods and equipment. Such
modifications will become apparent to those skilled in the art from
a consideration of this disclosure or practice of the invention
disclosed herein. Consequently, it is not intended that this
invention be limited to the specific embodiments disclosed herein,
but that it cover all modifications and alternatives coming within
the true scope and spirit of the invention.
Sequence CWU 1
1
1115DNAArtificial Sequencesynthetic oligonucleotide 1cttcattttt
tcttc 15
* * * * *